Discussion The results of these studies indicate that immunocompetent SP-A KO mice, but not WT mice, harbor viable and actively replicating P. murina following environmental exposure. The source of Pneumocystis spp. infection in humans and animals remains unknown. Various species have been detected by environmental sampling techniques, suggesting a possible environmental source of the infection; yet, the infectious potential of these environmental samples has not been demonstrated [ 36- 39]. Human-to-human transmission of P. jirovecii has been identified by epidemiology studies that identified direct transmission and clusters of infections [ 40- 42]. Conversely, other studies determined that person-to-person transmission of P. jirovecii did not appear to contribute significantly to the spread of the disease [ 43, 44]. Several studies have demonstrated that recurrence of PcP following successful treatment involves P. jirovecii of a different strain from the initial infection, suggesting that the recurrence is the result of infection from an exogenous source that may be either due to environmental exposure or person-to-person transmission [ 45, 46]. The development of sensitive PCR techniques led to the search for the presence of low levels P. jirovecii in the lungs of immunocompetent and immunocompromised humans [ 47]. It had long been thought Pneumocystis remained in a latent stage within the lungs of immunocompetent individuals and became reactivated during immunosuppression; however, results from early studies indicated that P. jirovecii was not present in lungs of non-immunosuppressed individuals [ 48- 50]. In addition, a more recent study also indicated that healthy subjects may not be colonized with P. jirovecii [ 51]. These results suggest that P. jirovecii does not exist in a latent stage in immunocompetent individuals. Although, P. jirovecii colonization was not detected in healthy individuals, P. jirovecii colonization has been described in some hospitalized patients with moderate to severe underlying immunodeficiency suggesting that this patient population could serve as a reservoir of infection [ 52]. Results from a recent study suggest that P. jirovecii colonization in HIV + hospitalized patients may also be a source of infection. In that study, it was found that 68% of hospitalized HIV + patients with non PcP pneumonia were colonized with P. jirovecii [ 53]. In another recent study, P. jirovecii colonization was detected in 46% of the autopsy specimens of HIV+ individuals [ 7]. Also, in that study, cigarette smoking and city of residence were discovered to be risk factors for colonization. In a study, examining bronchoalveolar specimens, a lower level of colonization was detected in persons infected with HIV, but these authors also propose that this carriage may be a reservoir of infection [ 8]. P jirovecii colonization in patients with other lung diseases such as cystic fibrosis and chronic obstruction pulmonary disease, without significant underlying immunosuppression, has also been described [ 54]. The findings of an early study indicated that P. jirovecii colonization was not associated with cystic fibrosis. [ 55]. However, in two more recent studies colonization rates of 7.4% and 21.5% were detected in individuals with cystic fibrosis [ 56, 57]. Interestingly, it has also been show that P. jirovecii colonization in cystic fibrosis patients is a dynamic process, in which clearance and recolonization occurs over time [ 58]. Several reports of P. jirovecii colonization in patients with chronic obstructive pulmonary disease (COPD) have been published. In an early study, a colonization rate of only 10% was detected and the authors postulated that since this rate of colonization was similar to the rate of PcP being seen in immunocompromised patients at their site, PcP arose from a latent infection and was not the result of a new infection [ 59]. P. jirovecii colonization has also been linked with severity of COPD as measured by spirometric lung function in a study that examined autopsy specimens. In that study, colonization was detected in 37% of smokers with severe COPD, but only 5.3% of those with less severe disease [ 11]. P. jirovecii colonization in persons with COPD has also been associated with higher levels proinflammatory cytokines, suggesting that the host mounts an immune response to the colonization [ 12]. In this study, colonization was detected in 55% of induced sputum samples from COPD patients, a much higher rate of colonization than previously reported. The variation in reported colonization rates may be related to the type of samples that were tested or due to differences in the detection methods that were used. Alternatively, the different colonization rates may indicate that some demographic areas may have higher rates of colonization than others, as was reported in colonization rates in HIV+ individuals [ 7]. Animal-to-animal spread of the infection has been well documented and supports the role of immunocompetent hosts as reservoirs of Pneumocystis spp. infection. In studies using mouse models, transmission of P. murina infection through cohousing with infected mice initiates infection in both immunocompetent and immunocompromised animals [ 60, 61]. In normal mice, the colonization is self-limiting; immune-mediated clearance occurs 5–6 weeks post initiation of infection and involves both humoral and cellular immune responses [ 5, 62]. Normal mice with this transient colonization have also been shown to be able to transmit the infection, further supporting the normal host as a reservoir of infectious organisms [ 60, 62]. It was surprising to detect environmental exposure of P. murina within our barrier animal facility; however, this finding may not be totally unexpected. Previously, both P. murina and P. carinii outbreaks have been described in commercial animal facilities, demonstrating the ability of Pneumocystis to circumvent isolation systems designed to limit cage-to-cage exposure. It has been shown that air may circulate into cages with micro isolator tops not through the filter on top of the cage, but rather through the edges of the cage which may not be rigorously sealed [ 63]. Even though cages are changed within a laminar flow hood, another source of potential environmental exposure to P. murina would be during cage changing procedures. Cages of mice used in these studies were changed 2–3 times per week which may have lead to increased environmental exposure and development of colonization in the KO mice. This could result in cross-contamination of cages within the colony. Colonization is a very common finding in immunocompetent rat colonies that have been screened by PCR, but it usually goes undetected [ 64]. A systematic survey of normal mouse colonies has not been performed, but both colonization and active P. murina infection in several genetically immunodeficient mouse colonies has been described [ 65, 66]. These findings demonstrate that colonization of laboratory animals occurs in spite of maintenance of strict barrier conditions, and suggests that these practices are not sufficient to prevent the development of Pneumocystis colonization [ 66, 67]. In the present report, both WT and KO mice apparently encountered P. murina through environmental exposure within the animal facility, based on the development of P. murina specific serum antibodies in both strains of mice. In WT mice, it seems that the P. murina is eliminated from the lungs and never establishes an infection; yet, in KO mice the P. murina establishes a transient colonization. Based on these results, we propose that there are two potential outcomes of environmental exposure to Pneumocystis in the immunocompetent host: 1) Development of transient colonization. In this scenario, Pneumocystis escapes detection by the innate immune response and establishes a colonization that may be eventually cleared by the adaptive immune response. 2) Elimination of Pneumocystis without development of colonization. In this situation, the innate immune response recognizes and eliminates the Pneumocystis prior to it being able to establish an active infection. It is likely that the level of environmental exposure within the animal facility is very low and sporadic. WT mice appear to be able to defend against such a low level exposure; however, the absence of SP-A in the KO allows the P. murina to escape innate host responses, which are likely to be involved in protection of the host from initial infection. It is interesting to note that at the time that these studies were performed at the UC LAM facility, colonization was not detected in SCID mice housed under the same conditions in microisolator cages. It was thought that the absence of colonization in the SCID mice reflected the presence of SP-A and/or residual immune function. Recently, our mouse colonies have been relocated to a different animal facility. The housing conditions in this facility are similar to conditions used in the previous facility, but sporadic colonization has been observed in SCID mice housed in microisolator cages in this facility. These new findings indicate that SCID mice are susceptible to development of colonization following environmental exposure, and support the idea that all of the mice within the facility are environmentally exposed to P. murina. These data also emphasize the complexities of developing animal models of Pneumocystis colonization. On the one hand, acquisition of P. murina colonization via natural environmental exposure most closely mimics the situation in humans, as other methods of transmitting P. murina such as intratracheal inoculation, intranasal inoculation, or co-housing infected with uninfected mice ("seeding") involve larger or more intense types of exposure. On the other hand, environmental exposure studies are uncontrolled and become difficult to interpret when P. murina infection already exists in some members of an animal colony. Although immunocompetent SP-A KO mice are susceptible to P. murina colonization, they can limit and ultimately clear the infection. Not surprisingly, CD4+ T cells appear to play an important role in this process. Increased numbers of CD4+ T-cells were detected in the KO mice compared to the WT mice, and antibody mediated CD4+ T-cells depletion resulted in higher levels of infection in the KO mice. Further characterization of the SP-A independent factors involved in clearance of colonization will provide insight into the mechanisms immunocompetent hosts utilize to defend against Pneumocystis infection. SP-A is a member of the collectin family of proteins, innate immune molecules containing a collagen-like amino terminus and a C-type lectin carbohydrate recognition domain at the carboxyl terminus [ 15]. Structurally similar to C1q, mannose-binding protein and surfactant protein D (SP-D), it is secreted by type II alveolar epithelial cells. SP-A KO mice have provided valuable information on the role of SP-A in host defenses against P. murina and other organisms [ 17]. These animals have normal lung morphology, physiology, surfactant phospholipids and other surfactant proteins (SP-B, SP-C or SP-D), but are deficient in tubular myelin [ 16]. KO mice have impaired clearance of infectious agents following experimental exposure, which is usually accompanied by increased proinflammatory cytokines [ 17]. A recent study demonstrated that SP-A null mice raised in an environment heavily contaminated with bacteria were more susceptible to bacterial peritonitis than WT mice [ 68]. Although the lungs were not involved in the infection in this model, the results demonstrate that SP-A KO mice may also be susceptible to infections following environmental exposure. It has been previously shown that immunosuppressed SP-A KO mice are more susceptible to P. murina infection, and when immunosuppressed, they develop higher levels of PcP than WT mice [ 24, 25]. Immunocompetent KO mice also display delayed clearance following infection by intratracheal inoculation compared to WT mice [ 69]. However, SP-A did not enhance clearance of P. murina in a steroid treated mouse model of infection. In this model, KO mice cleared the infection just as efficiently as WT mice following withdrawal of the corticosteroid induced immunosuppression [ 34]. SP-A plays a role in early host defenses by enhancing the uptake of infectious agents by alveolar macrophages and has been shown to facilitate the adherence and/or phagocytosis of most of the organisms with which it has been tested[ 70]. In vitro studies have demonstrated that under certain conditions SP-A enhances attachment of Pneumocystis to AMs; however, inhibition of Pneumocystis binding to alveolar macrophages by SP-A has also been reported [ 71, 72]. In our laboratory, preincubation of P. murina, which was isolated from KO mice, with SP-A resulted in enhanced attachment to mouse alveolar macrophages [ 24]. SP-A also displays other protective activities in the host response. Binding of SP-A to gram-negative bacteria, Mycoplasma pneumoniae, and Histoplasma capsulatum has been shown to result in killing of the organisms [ 23]. SP-A also increases aggregation of organisms that leads to enhanced clearance from the respiratory tract by mucociliary actions [ 17]. In addition to these direct activities, SP-A interacts with a variety of immune cells and modulates their responses to pulmonary pathogens [ 15]. SP-A has been shown to inhibit maturation of dendritic cells and limit their ability to activate T cells [ 73]. In addition to its regulation of the innate immune response, there is rising interest in the interaction of SP-A with the adaptive immune system [ 74]. A recent report showed that SP-A binds to the Fc portion of IgG and facilitates the uptake of IgG coated erythrocytes by AMs [ 75]. These other SP-A activities have not been characterized in the host response to Pneumocystis; however, it is likely that prevention of colonization involves both innate and adaptive immune response mechanisms and that SP-A may have additional activities beyond enhancing uptake by alveolar macrophages. The current study demonstrates a new role for SP-A in protecting the immunocompetent host from P. murina infection by preventing the normal host from becoming infected with P. murina following environmental exposure. The protective effects of SP-A in this situation, may involve direct action of the molecule on P. murina viability, indirect modulation of other factors of the host immune response, or enhanced recognition of P. murina by alveolar macrophages [ 23, 23, 72, 76]. SP-A could also exert its protective effects by a combination of all these activities. Pneumocystis colonization in immunocompetent hosts may be a mechanism that the organism uses as a survival and transmission strategy, thus is relevant to the care of immunocompromised patients. The detection of significant P. jirovecii colonization rates in humans suggests the need for caution in interactions between hospital staff and immunocompromised patients [ 77]. Identification of factors involved in protecting the immunocompetent host from becoming infected with Pneumocystis, may provide tools to eliminate this reservoir of infection and reduce spread of the disease to highly susceptible immunocompromised patients. A role for SP-A in host defense against P. murina infection has been clearly demonstrated in KO mouse models and by in vitro experiments [ 24, 26]. However, the role of SP-A in the host response to P. jirovecii infection in humans has not yet been established. It has been shown that immunodeficient individuals with HIV/AIDS and PcP have increased levels of SP-A in BALF compared to individuals with bacterial pneumonias [ 78]. In that work, the authors did not demonstrate a functional role for SP-A in the host response to P. jirovecii in humans, but the results suggest that SP-A may be involved. The results presented in the current study indicate that SP-A may also be involved in protecting the immunocompetent host from Pneumocystis colonization. As described above, the pathophysiologic mechanisms associated with P. jirovecii colonization in humans, in addition to immunodeficiency, include chronic pulmonary diseases and smoking [ 79]. In humans, SP-A levels within BALF have been show to vary considerably among normal individuals and also among persons with various lung diseases [ 80]. It is interesting to note that SP-A levels have been shown to be decreased by smoking and smoking has also been identified as a risk factor for P. jirovecii colonization. Recent work has also demonstrated that exposure to cigarette smoke leads to increased burdens of P. murina in mice [ 81]. Two unique variants of SP-A (SP-A1 and SP-A2) are expressed from two different genes in humans and more than 30 alleles have been described for the SP-A genes [ 82]. It has been shown that the SP-A variants have functional differences and postulated that SP-A2 may be more active in the innate immune response than SP-A1 [ 83, 84]. Based on these findings it is possible that decreased SP-A levels or expression of particular SP-A variants may lead to enhanced susceptibility to P. jirovecii colonization in humans. |
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