Veterinary Immunology and Immunopathology 163 (2015) 1–7
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Research paper
Local pulmonary immune responses in domestic cats naturally infected with Cytauxzoon felis Karelma Frontera-Acevedo a , Kaori Sakamoto b,∗ a School of Veterinary Medicine, Faculty of Medical Sciences, University of the West Indies, Building 47, Eric Williams Medical Sciences Complex, Uriah Butler Highway, Champ Fleurs, Trinidad and Tobago b Department of Pathology, The University of Georgia, College of Veterinary Medicine, 501 D.W. Brooks Dr., Athens, GA 30602, United States
a r t i c l e
i n f o
Article history: Received 2 July 2014 Received in revised form 10 October 2014 Accepted 29 October 2014 Keywords: Cat Cytauxzoon felis Immune response Lung Macrophage Pathology
a b s t r a c t Cytauxzoonosis is a hemoprotozoal disease of cats and wild felids in the South and Southeastern United States caused by Cytauxzoon felis. Although the causative agent has been recognized since the seventies, no study has examined the local immune response in affected organs, such as the lung, and compared them to the lungs of uninfected domestic cats. Previous studies have suggested that the histopathologic findings in the lungs of C. felis-infected cats are caused by the release of pro-inflammatory mediators, such as cytokines and increased production of inducible nitric oxide synthase (iNOS), by the infected macrophages. Our laboratory had previously found an upregulation of the adhesion molecule CD18, which can stimulate the release of these pro-inflammatory mediators. The objective of this study was to characterize local pulmonary immune responses in cats naturally infected with C. felis. Immunohistochemistry was performed to detect tumor necrosis factor-␣ (TNF-␣), interleukin (IL)-1, IL-6, iNOS, and major histocompatibility complex (MHC) II in 19 lungs from affected cats that died between 2005 and 2013. Results showed increased expression of all of these molecules when compared to lungs from uninfected, healthy cats. Furthermore, MHC II is expressed in the endothelium of C. felis naturally infected cats. These results support that there is a marked, local, pro-inflammatory immune response that can contribute to the pathogenesis of cytauxzoonosis in the lungs. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Cytauxzoonosis is a fatal disease of domestic cats in the Midwestern, Mid-Atlantic, Southeastern, and Southcentral United States, caused by Cytauxzoon felis, a tick-borne parasite in the order Piroplasmida, family Theileriidae. An infected tick transmits C. felis while feeding, followed by schizogeny of the parasite in monocytes/macrophages
Abbreviation: iNOS, inducible nitric oxide synthase. ∗ Corresponding author. Tel.: +17065425844; fax: +706 542 5828. E-mail address: [email protected] (K. Sakamoto). http://dx.doi.org/10.1016/j.vetimm.2014.10.012 0165-2427/© 2014 Elsevier B.V. All rights reserved.
throughout the body (Fry and McGavin, 2012). While this infection can be asymptomatic, clinical signs in infected cats include anorexia, depression, lethargy, dehydration, pyrexia, dyspnea, icterus, dark urine, and less commonly, pallor, anemic heart murmur, and increased capillary refill time. Hematologic findings may include normocytic, normochromic, nonregenerative anemia, with pancytopenia or moderate neutrophilia (Greene et al., 2006; Reichard et al., 2009). Since its discovery and description in the 1970s, very little has been published regarding the feline immune response to this disease. A previous study reported the formation of antibodies against the non-pathogenic,
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erythrocytic stage of C. felis (Cowell et al., 1988). Kier et al. (1987) confirmed the monocyte/macrophage identity of the infected cells in the leukocytic stage of the disease. Snider et al. (2010) described and categorized the interstitial pneumonia commonly present in cats that died from C. felis infection and suggested that this inflammation is likely caused by release of pro-inflammatory cytokines and chemokines by the infected macrophages. One of the main histopathologic characteristics of cytauxzoonosis is the presence of giant, infected, intravascular monocyte/macrophages, many of which are adhered to the vascular endothelium, with activation and involvement of CD18. Our laboratory had previously found an upregulation of this molecule (Frontera-Acevedo et al., 2013), which can stimulate the release of some pro-inflammatory mediators, such as TNF-␣, IL-1, IL-6, and inducible nitric oxide synthase (iNOS). That paper characterized the systemic immune response to C. felis infection, and supported the hypothesis that cytauxzoonosis causes a robust systemic pro-inflammatory response characterized by increases in pro-inflammatory cytokines, an acute-phase response, IgM deposition on erythrocytes, and upregulation and expression of CD18 on leukocytes. This response is more severe in cats that died of C. felis infection, compared with the response in cats that survived the disease, which suggests that the immune response is important in the pathogenesis of this disease. Immunohistochemical characterization of the local pulmonary immune response can demonstrate the effects of the immune response in one of the main tissues in which pathologic changes due to C. felis infection have been recognized, and which contributes to the high morbidity and mortality of this disease. 2. Materials and methods 2.1. Case selection Nineteen samples of lung tissues from cats that died of cytauxzoonosis and were received for necropsy at the Athens Diagnostic Laboratory between the years 2005 and 2013 were selected for further immunohistochemical studies. For all of the cases selected, the original hematoxylin and eosin slides containing lung tissue were examined by a board-certified, veterinary pathologist. For negative controls, one archived research case from a healthy control cat used in another experiment was used. For comparison purposes, two cats that died of conditions unrelated to cytauxzoonosis or pulmonary disease (hemolytic anemia and cholangiohepatitis) were also examined. 2.2. TNF-˛, IL-1ˇ, IL-6, iNOS, and MHC II immunohistochemistry
CA), mouse anti-feline IL-6 monoclonal antibody (Clone 341031, RnD Systems, Minneapolis, MN), goat anti-feline IL-1 polyclonal antibody (Catalog Number AF1796, RnD Systems, Minneapolis, MN), mouse anti-human HLA-DR, ␣-chain monoclonal antibody (Clone TAL 1B5, Santa Cruz Biotechnology, Santa Cruz, CA), and rabbit anti-mouse iNOS polyclonal antibody (Catalog Number PA3-030A, Thermo Scientific, Rockford, IL), and their appropriate isotype control antibodies (RnD Systems, Minneapolis, MN, AbD Serotec, Raleigh, NC). The TNF-␣, HLA-DR, and iNOS antibodies have been found to cross-react in cats (Fernandez et al., 2008; Islam et al., 2012; Pullen et al., 1997; Roosje et al., 1997). Two different kits were used for stain development (Dako, Carpinteria, CA; RnD Systems, Minneapolis, MN), based on published protocols and manufacturer’s recommendations. Each slide was incubated with primary antibody either overnight at 4 ◦ C (TNF-␣, IL-1, IL-6, iNOS), or at room temperature for 3 h (MHC II). Slides were counterstained with Gill’s hematoxylin stain and rinsed with deionized water, and a coverslip was then added. More details about these procedures are presented in Table 1. Staining distribution was subjectively compared via light microscopy by a board-certified veterinary pathologist. Immunostained slides were examined in one session per molecule to reduce variability, and a repeat analysis was performed to confirm reproducibility prior to statistical analysis. The TNF-␣ immunoreactivity was characterized as low, moderate, or high. For TNF-␣, low staining was defined as extremely light, diffuse, background staining (but more staining than for negative control samples) and no cytoplasmic staining in the inflammatory cells. Moderate staining was defined as diffuse TNF-␣ immunoreactivity evident in the cytoplasm of less than half of the inflammatory (mostly infected) cells. High staining was defined as staining evident in more than half of the inflammatory cells. The IL-1, IL-6, and iNOS immunoreactivity was characterized as low or high. For both cytokines and iNOS, low staining was defined as extremely light, diffuse, background staining (but more staining than for negative control samples) and scattered intracytoplasmic staining in few scattered alveolar macrophages. High staining was characterized as more diffuse intracytoplasmic staining present in at least half of the various cell types examined. The MHC II immunoreactivity was characterized as low, moderate, or high. For MHC II, low staining was defined as staining of only the cell membrane of a few alveolar macrophages. Moderate staining was defined as either staining of cells besides alveolar macrophages or plasmalemmal staining of the endothelium, but not both present at the same time. High staining was defined as staining of both leukocytes and the endothelium. 2.3. Statistical analysis
Tissues were fixed in 10% neutral-buffered formalin, embedded in paraffin, cut at a thickness of 4 m, and placed on glass slides. If antigen retrieval was performed, a 10 mM sodium citrate solution (pH 6.0) was used. Antibodies used included: goat anti-mouse TNF-␣ polyclonal antibody (Clone M-18, Santa Cruz Biotechnology, Santa Cruz,
For immunohistochemical analysis, a Fischer’s exact test (InStat, Graphpad Software, La Jolla, CA) was performed to determine significant differences between infected and uninfected groups in terms of expression of TNF-␣, IL-1, IL-6, iNOS, and MHC II.
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Table 1 Primary antibodies, dilutions, and antigen retrieval methods used. N/A = no antigen retrieval used, ABC = avidin–biotin complex kit, Polymer = polymer/streptavidin kit. Primary antibody
Dilution
Final concentration (g/mL)
Antigen retrieval method
Detection method
TNF-␣ IL-1 IL-6 iNOS MHCII
1:100 1:10 1:10 1:100 1:50
N/A 10 50 N/A 4
L.A.B Citrate N/A Citrate Citrate
Polymer ABC ABC ABC Polymer
3. Results 3.1. Histopathology results The major pulmonary histopathologic findings are summarized in Table 2. Of the nineteen cases examined, four lungs were diagnosed by the anatomic pathologist who performed the necropsy as pneumonia (three interstitial pneumonias, one bronchopneumonia), and three were diagnosed with vasculitis, not just limited to the lung lesions. Fig. 1 exemplifies all of the major histopathologic findings present. 3.2. Pro-inflammatory cytokines and iNOS immunohistochemistry All lung tissues obtained from cats that died of C. felis stained positively for TNF-␣ by immunohistochemistry. The tissues were characterized as having high staining, particularly in the cytoplasm of infected macrophages that lined vessels and other inflammatory cells within the pulmonary interstitium and alveoli (Fig. 2A). Of the 19 lung tissues from cats that died of C felis, 16 had moderate staining for IL-1 by immunohistochemistry, particularly in the cytoplasm of infected cells that lined vessels and other inflammatory cells within the pulmonary interstitium and alveoli (Fig. 3A). Occasional staining was also noted in the cytoplasm of endothelial cells and surrounding alveolar macrophages. The lung tissues from the three uninfected cats had low staining for IL-1, and in
some cases, complete absence of staining (Fig. 3C). A significant difference (P = 0.013) in staining for IL-1 was observed between cats that died of cytauxzoonosis and the uninfected cats. Of the 19 lung tissues from infected cats, 16 had moderate staining for IL-6 by immunohistochemistry, particularly in the cytoplasm of infected cells and uninfected monocytes, alveolar macrophages, lymphocytes, and neutrophils, with rare intracytoplasmic staining of the vascular endothelium (Fig. 4A). Only one case from an infected cat had low immunoreactivity for both IL-1 and IL-6; another two cases that had low immunoreactivity for IL-6 did had high immunoreactivity for IL-1. The lung tissues from the three uninfected cats also had low or absent staining for IL6 (Fig. 4C). A statistically significant difference (P = 0.013) in staining for IL-6 was observed between cats that died of cytauxzoonosis and the uninfected cats.
3.3. iNOS immunohistochemistry Fourteen lung tissues from infected cats had moderate immunoreactivity for iNOS, while only five lung tissues from infected cats had low immunoreactivity. All tissues from uninfected cats had low immunoreactivity to iNOS. The staining was characterized by cytoplasmic staining in the infected cells, as well as in the surrounding monocytes and alveolar macrophages (Fig. 5). One case that had low staining for IL-6 also had low staining for iNOS, but the other four cases that had low staining for iNOS had moderate staining for both IL-1 and IL-6. A significant difference (P = 0.036) in staining for iNOS was observed between C. felis-infected cats and uninfected cats.
3.4. MHC II immunohistochemistry
Fig. 1. Typical histopathologic appearance of C. felis-infected lung. Note the enlarged, intravascular, infected monocytes, interstitial pneumonia, and alveolar edema.
Immunohistochemistry for MHC II revealed that expression was extremely low in all uninfected cats (Fig. 6C) and was restricted to cytoplasmic staining of a few scattered interstitial macrophages. Cats that died of C. felis infection had moderate (3) to high (16) MHC II expression, as indicated by cytoplasmic staining for MHC II in various cells (Fig. 6A), including in the endothelium. Only three lung tissues from cats that died of cytauxzoonosis did not demonstrate staining of infected cells, but had MHC II cytoplasmic immunoreactivity in the endothelium. A significant difference (P = 0.0006) in staining for MHC II was observed between the cats that died of the disease and uninfected cats.
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Fig. 2. Immunohistochemistry for TNF-␣. (A) Section of lung from a C. felis-infected cat, using anti-TNF-␣ antibody. Note high intensity of immunoreactivity, particularly within the cytoplasm of infected, intravascular monocytes and interstitial leukocytes. (B) Section of lung from a C. felis-infected cat, using isotype antibody. Note background staining of leukocytes, most likely due to endogenous peroxidases. (C) Section of lung from an uninfected cat, using anti-TNF-␣ antibody. Note occasional immunoreactive interstitial leukocytes.
Fig. 3. Immunohistochemistry for IL-1. (A) Section of lung from a C. felis-infected cat, using anti-IL-1 antibody. Note high intensity of immunoreactivity, particularly within the cytoplasm of infected, intravascular monocytes and interstitial leukocytes. (B) Section of lung from a C. felis-infected cat, using isotype antibody. Note slight background staining of infected, intravascular monocytes. (C) Section of lung from an uninfected cat, using anti-IL-1 antibody. Note absence of immunoreactivity.
Fig. 4. Immunohistochemistry for IL-6. (A) Section of lung from a C. felis-infected cat, using anti-IL-6 antibody. Note high intensity of immunoreactivity, particularly within the cytoplasm of infected, intravascular monocytes. (B) Section of lung from a C. felis-infected cat, using isotype antibody. Note slight background staining of infected, intravascular monocytes and interstitial leukocytes. (C) Section of lung from an uninfected cat, using anti-IL-1 antibody. Note absence of immunoreactivity.
Fig. 5. Immunohistochemistry for iNOS. (A) Section of lung from a C. felis-infected cat, using anti-iNOS antibody. Note high intensity of immunoreactivity, particularly within the cytoplasm of infected, intravascular monocytes. (B) Section of lung from a C. felis-infected cat, using isotype antibody. Note absence of immunoreactivity. (C) Section of lung from an uninfected cat, using anti-iNOS antibody. Note absence of immunoreactivity.
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Table 2 Distribution of major pulmonary histopathologic findings in C. felis-infected cats. Numbers are out of a total of 19 C. felis-infected cases that were selected for immunohistochemistry. Major pulmonary histopathologic findings Edema
Alveolar hemorrhage
Thickened septa
Neutrophils within septa
Alveolar macrophages
19
12
11
9
16
4. Discussion Although a complete detailed analysis of histopathologic findings was not performed as described by Snider et al. (2010), a combination of the main significant findings he observed was present in all of our cases. The pulmonary lesions in six of the cases studied were originally reported as interstitial pneumonia or vasculitis. Characteristics of interstitial pneumonia included edema, thickening of the interstitium by edema and neutrophilic infiltrate, and alveolar neutrophilic exudate, among others (López, 2012). At least two of these lesions were present in all lung sections examined, supporting that infection with C. felis causes an interstitial pneumonia. There were also three cases that were interpreted to have vasculitis, an inflammatory lesion that can be a component of systemic disease (Snyder, 2012), such as C. felis infection. Characteristics of vasculitis include the presence of leukocytes within and around the affected vessels, as well as damage to the vessel walls, usually characterized by necrosis or fibrin deposits (Miller et al., 2012). In the cases examined, although necrosis was not noted and fibrin was rare, the common presence of alveolar hemorrhage implies that there had been damage to the vessel walls. In addition, in many cases, neutrophils were observed surrounding the small- to medium-caliber vessels and the alveolar capillaries. Immunohistochemistry of C. felis – infected tissue samples revealed a significant, widespread, qualitative increase in the expression of the pro-inflammatory cytokines TNF␣, IL-1, and IL-6, as well as iNOS, compared with results from uninfected tissues. Detection of these molecules in uninfected control cats was either completely absent or very limited and scattered. A study characterizing the morphologic features of feline infectious peritonitis (FIP) (Kipar et al., 2005) described similar increases in the proinflammatory cytokines TNF-␣ and IL-1 in the tissues of
FIP-infected cats, and related this expression to upregulation by CD18, which has also been suggested by other studies (Kipar et al., 2005; Lee et al., 2000; Leite et al., 2003), as well as our previous study on systemic immune responses to C. felis infection (Frontera-Acevedo et al., 2013). That study found not only a qualitative increase in CD18 expression, but also a relative quantitative increase in CD18 transcription in cats that died of C. felis infection compared to uninfected cats; therefore, the qualitative increase in the pro-inflammatory cytokines observed in this study may be related to the increase in the expression of CD18. The occasional immunoreactivity for pro-inflammatory cytokines noted on the endothelium was also described in the vessels of FIP-infected cats (Kipar et al., 2005), and this was explained in that article as a local paracrine effect of the infected macrophages on the endothelium, instead of a systemic response. Unlike the other two cytokines, IL-6 was found to be less disseminated, perhaps because IL-6, although still part of the innate immune response, is produced slightly later than the other two cytokines (Frontera-Acevedo et al., 2013). Although no previous study has identified the expression of TNF-␣, IL-1, and IL-6 in tissues from C. felis-infected cats, expression of all of these cytokines has been found to be increased in cell lines infected with Theileria annulata (Brown et al., 1995; McGuire et al., 2004). A previous study on C. felis has already addressed the possibility of proinflammatory cytokines being involved in the pathogenesis of the pulmonary lesions (Snider et al., 2010), and the present immunohistochemistry study confirms the extensive presence of TNF-␣, IL-1, and IL-6 in the lung. The strong iNOS immunoreactivity present in the infected cases demonstrates that the infected macrophages are likely actively producing reactive nitrogen intermediates, which is absent in resting macrophages and monocytes, but is induced in response to activation
Fig. 6. Immunohistochemistry for MHC II. (A) Section of lung from a C. felis-infected cat, using anti-MHC II antibody. Note high intensity of immunoreactivity, particularly within the cytoplasm of infected, intravascular monocytes. Inset: High magnification of endothelium from another case showing strong cytoplasmic immunoreactivity of endothelial cells. (B) Section of lung from a C. felis-infected cat, using isotype antibody. Note absence of immunoreactivity. (C) Section of lung from an uninfected cat, using anti-iNOS antibody. Note absence of immunoreactivity.
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(Abbas et al., 2010). This combined expression of proinflammatory cytokines and iNOS in infected macrophages demonstrates that they are activated, as was suggested in a previous study (Snider et al., 2010), and this contributes to the histologic lesions, morbidity, and mortality characteristically observed with this disease. iNOS mRNA expression has been found to be increased in Theileria parva infection (Okagawa et al., 2012), but decreased expression of NO and iNOS has been shown in a study of T. annulata-infected macrophages (Sager et al., 1997). The expression of pro-inflammatory cytokines (TNF-␣, IL-1, IL6) and iNOS is an immunohistochemical feature also found in what is considered acute pulmonary distress syndrome (ARDS), both in humans and animals (López, 2012; Tsushima et al., 2009). Snider et al. (2010) had suggested that cats that died from cytauxzoonosis may be affected with ARDS, based on the histopathologic findings. ARDS is a clinical diagnosis that, in veterinary medicine, has been defined as a condition meeting four of the following five criteria: (1) acute onset of tachypnea and difficulty breathing, (2) presence of risk factors (which includes inflammation, sepsis, or infection), (3) evidence of pulmonary capillary leakage without increased pulmonary capillary hydrostatic pressure, (4) inefficient gas exchange, and (5) evidence of diffuse pulmonary inflammation (López, 2012; Wilkins et al., 2007). One of the main features of ARDS is the increase in alveolar septal and vascular permeability, something that was noted in both Snider’s et al. (2010) study and the present study. One histopathologic feature, though, that is commonly found in human cases of ARDS, but was rarely found by either Snider or this study, is the presence of hyaline membranes lining the alveolar septa (Matthay and Zemans, 2011). Although ARDS is usually caused by sepsis, it is also on occasion caused by a parasitic infection, such as malaria in humans, which is caused by various Plasmodium spp. (Autino et al., 2012; Bauer et al., 2006). In those cases, parasite sequestration in the vascular endothelium causes release of pro-inflammatory mediators (TNF-␣, IL-1, IL-6, NO) and resultant tissue damage. The immunohistochemical surface detection of MHC II was also increased in C. felis-infected lung samples compared to uninfected lungs. This is not only due to the marked increase of antigen-presenting cells in the infected tissue, but also in the expression of MHC II by the endothelium. Endothelial cells are not cell types associated with constitutive MHC II expression, but MHC II expression can be activated and induced by immune mediators, such as IFN-␥ or TNF-␣ in various species (Abbas et al., 2010; Batten et al., 1996; Kariuki Njenga and Dangler, 1995). We recently showed a marked increase in TNF-␣ concentrations serologically in C. felis-infected cats (Frontera-Acevedo et al., 2013), and it is possible that this cytokine was responsible for the induction of MHC II expression in endothelial cells. Unlike endothelial expression of pro-inflammatory cytokines and other molecules, endothelial expression of MHC II happened independently of the presence of nearby infected (and immunoreactive) macrophages. In three cases, the infected macrophages failed to stain for MHC II, but had marked endothelial MHC II immunoreactivity. This endothelial MHC II expression
was considered a systemic response in a study of FIPinfected cats (Kipar et al., 2005), unlike the endothelial expression of pro-inflammatory cytokines. Activated endothelium can also modulate inflammatory responses and can contract (Ackermann, 2012), allowing fluid to leak into the extravascular space and resulting in the edema that was noted in the lungs histologically in this and other studies (Snider et al., 2010). They can also stimulate production and secretion of NO, which although usually a vasodilator, can in large amounts increase inflammation and damage the surrounding tissues (Abbas et al., 2010; Ackermann, 2012). As noted by immunohistochemistry, the endothelial cells in this study did occasionally express iNOS, and the overexpression of NO by vascular walls is known to contribute to certain shock symptoms, such as hypotension and microvascular damage (Forstermann and Sessa, 2012). These effects can then cause congestion and edema, which are part of the common pathologic findings noted in C. felis-infected cats (Greene et al., 2006). A limitation of these immunohistochemical studies is that, while they helped to characterize the local immune response and confirm the presence of pro-inflammatory cytokines in the affected tissues, they cannot measure the local concentration of cytokines produced, or the relative increase in cytokine mRNA expression. In our previous study (2013), we were able to demonstrate a marked increase in the serum concentrations of TNF-␣ and IL-1 in cats that had died of the disease compared to cats that survived, but it was limited in that the disease course was not uniform because the sample population consisted of naturally infected cats. The early studies that characterized cytauxzoonosis in cats by experimental infection described the gross and histologic characteristics of the disease (Kier et al., 1982a,b; Wagner et al., 1980), but at that time did not elucidate immunopathologic mechanisms. The lack of a C. felis in vitro culture system also limits the study of this disease without the infection of live cats.
5. Conclusions Overall, the findings in this paper confirm previous suggestions (Frontera-Acevedo et al., 2013; Snider et al., 2010) that the C. felis-infected macrophages are activated in a classical or M1 fashion, secreting and/or expressing various pro-inflammatory mediators. Although, typically, a M2 macrophage response is expected during parasite infections (Sica and Mantovani, 2012), it is possible that C. felis can somehow alter and modulate the response in the macrophage to ensure survival and replication. The resulting pro-inflammatory local and systemic immune responses are then responsible for the morbidity and mortality associated with this disease. Several articles have been published recently describing various methods of potentially preventing and treating cytauxzoonosis in cats (Lewis et al., 2014; Reichard et al., 2013; Schreeg et al., 2013; Tarigo et al., 2013), but none of them have studied the pathogenesis of the disease or the role of the feline immune system. The findings in this article can be useful in directing the development of new, improved treatments for cytauxzoonosis.
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Conflict of interest The authors report no conflict of interest in the preparation of this manuscript. Acknowledgements The authors would like to thank the Athens Diagnostic Veterinary Laboratory in lending the archived blocks (pre-2009) and the University of Georgia Histology Laboratory for preparing them for immunohistochemistry. The authors also appreciate the help of the Georgia and Arkansas veterinarians who participated in the project between the years 2009 and 2012 and provided the cases for those years. References Abbas, A.K., Lichtman, A.H., Pillai, S., 2010. Cellular and Molecular Immunology, 6th ed. Saunders/Elsevier, Philadelphia, pp. 566. Ackermann, M.R., 2012. Inflammation and healing. In: Zachary, J.F., McGavin, M.D. (Eds.), Pathologic Basis of Veterinary Disease. , 5th ed. Elsevier, St. Louis, MO, pp. 89–146. Autino, B., Corbett, Y., Castelli, F., Taramelli, D., 2012. Pathogenesis of malaria in tissues and blood. Mediter. J. Hematol. Infect. Dis. 4, e2012061. Batten, P., Yacoub, M.H., Rose, M.L., 1996. Effect of human cytokines (IFN-gamma, TNF-alpha, IL-1 beta, IL-4) on porcine endothelial cells: induction of MHC and adhesion molecules and functional significance of these changes. Immunology 87, 127–133. Bauer, T.T., Ewig, S., Rodloff, A.C., Muller, E.E., 2006. Acute respiratory distress syndrome and pneumonia: a comprehensive review of clinical data. Clin. Infect. Dis. 43, 748–756. Brown, D.J., Campbell, J.D., Russell, G.C., Hopkins, J., Glass, E.J., 1995. T cell activation by Theileria annulata-infected macrophages correlates with cytokine production. Clin. Exp. Immunol. 102, 507–514. Cowell, R.L., Fox, J.C., Panciera, R.J., Tyler, R.D., 1988. Detection of anticytauxzoon antibodies in cats infected with a Cytauxzoon organism from bobcats. Vet. Parasitol. 28, 43–52. Fernandez, R., Gonzalez, S., Rey, S., Cortes, P.P., Maisey, K.R., Reyes, E.P., Larrain, C., Zapata, P., 2008. Lipopolysaccharide-induced carotid body inflammation in cats: functional manifestations, histopathology and involvement of tumour necrosis factor-alpha. Exp. Physiol. 93, 892–907. Forstermann, U., Sessa, W.C., 2012. Nitric oxide synthases: regulation and function. Eur. Heart J. 33, 829–837, 837a–837d. Frontera-Acevedo, K., Balsone, N.M., Dugan, M.A., Makemson, C.R., Sellers, L.B., Brown, H.M., Peterson, D.S., Creevy, K.E., Garner, B.C., Sakamoto, K., 2013. Systemic immune responses in Cytauxzoon felis-infected domestic cats. Am. J. Vet. Res. 74, 901–909. Fry, M.M., McGavin, D., 2012. Bone marrow, blood cells, and the lymphatic system. In: Zachary, J.F., McGavin, M.D. (Eds.), Pathologic Basis of Veterinary Disease. , 5th ed. Elsevier, St. Louis, MO, pp. 733–734. Greene, C.E., Meinkoth, J., Kocan, A.A., 2006. Cytauxzoonosis. In: Greene, C.E. (Ed.), Infectious Diseases of the Dog and Cat. , 3rd ed. Elsevier Saunders, Edinburgh, pp. 716–722. Islam, M.S., Matsumoto, M., Hidaka, R., Miyoshi, N., Yasuda, N., 2012. Expression of NOS and VEGF in feline mammary tumours and their correlation with angiogenesis. Vet. J. 192, 338–344. Kariuki Njenga, M., Dangler, C.A., 1995. Endothelial MHC class II antigen expression and endarteritis associated with Marek’s disease virus infection in chickens. Vet. Pathol. 32, 403–411. Kier, A.B., Wagner, J.E., Kinden, D.A., 1987. The pathology of experimental cytauxzoonosis. J. Comp. Pathol. 97, 415–432. Kier, A.B., Wagner, J.E., Morehouse, L.G., 1982a. Experimental transmission of Cytauxzoon felis from bobcats (Lynx rufus) to domestic cats (Felis domesticus). Am. J. Vet. Res. 43, 97–101. Kier, A.B., Wightman, S.R., Wagner, J.E., 1982b. Interspecies transmission of Cytauxzoon felis. Am. J. Vet. Res. 43, 102–105. Kipar, A., May, H., Menger, S., Weber, M., Leukert, W., Reinacher, M., 2005. Morphologic features and development of granulomatous vasculitis in feline infectious peritonitis. Vet. Pathol. 42, 321–330.
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Lee, H.Y., Kehrli Jr., M.E., Brogden, K.A., Gallup, J.M., Ackermann, M.R., 2000. Influence of beta(2)-integrin adhesion molecule expression and pulmonary infection with Pasteurella haemolytica on cytokine gene expression in cattle. Infect. Immun. 68, 4274–4281. Leite, F., Gyles, S., Atapattu, D., Maheswaran, S.K., Czuprynski, C.J., 2003. Prior exposure to Mannheimia haemolytica leukotoxin or LPS enhances beta(2)-integrin expression by bovine neutrophils and augments LKT cytotoxicity. Microb. Pathog. 34, 267–275. Lewis, K.M., Cohn, L.A., Marr, H.S., Birkenheuer, A.J., 2014. Failure of efficacy and adverse events associated with dose-intense diminazene diaceturate treatment of chronic Cytauxzoon felis infection in five cats. J. Feline Med. Surg. 16, 157–163. López, A., 2012. Respiratory system, mediastinum, and pleurae. In: Zachary, J.F., McGavin, M.D. (Eds.), Pathologic Basis of Veterinary Disease. , 5th ed. Elsevier, St. Louis, MO, pp. 458–538. Matthay, M.A., Zemans, R.L., 2011. The acute respiratory distress syndrome: pathogenesis and treatment. Annu. Rev. Pathol. 6, 147–163. McGuire, K., Manuja, A., Russell, G.C., Springbett, A., Craigmile, S.C., Nichani, A.K., Malhotra, D.V., Glass, E.J., 2004. Quantitative analysis of pro-inflammatory cytokine mRNA expression in Theileria annulatainfected cell lines derived from resistant and susceptible cattle. Vet. Immunol. Immunopathol. 99, 87–98. Miller, L.M., Vleet, J.F.V., Gal, A., 2012. Cardiovascular system and lymphatic vessels. In: Zachary, J.F., McGavin, M.D. (Eds.), Pathologic Basis of Veterinary Disease. , 5th ed. Elsevier, St. Louis, MO, pp. 539–588. Okagawa, T., Konnai, S., Mekata, H., Githaka, N., Suzuki, S., Kariuki, E., Gakuya, F., Kanduma, E., Shirai, T., Ikebuchi, R., Ikenaka, Y., Ishizuka, M., Murata, S., Ohashi, K., 2012. Transcriptional profiling of inflammatory cytokine genes in African buffaloes (Syncerus caffer) infected with Theileria parva. Vet. Immunol. Immunopathol. 148, 373–379. Pullen, A.H., Humphreys, P., Baxter, R.G., 1997. Comparative analysis of nitric oxide synthase immunoreactivity in the sacral spinal cord of the cat, macaque and human. J. Anat. 191 (Pt 2), 161–175. Reichard, M.V., Meinkoth, J.H., Edwards, A.C., Snider, T.A., Kocan, K.M., Blouin, E.F., Little, S.E., 2009. Transmission of Cytauxzoon felis to a domestic cat by Amblyomma americanum. Vet. Parasitol. 161, 110–115. Reichard, M.V., Thomas, J.E., Arther, R.G., Hostetler, J.A., Raetzel, K.L., Meinkoth, J.H., Little, S.E., 2013. Efficacy of an imidacloprid 10%/flumethrin 4.5% collar (Seresto(R), Bayer) for preventing the transmission of Cytauxzoon felis to domestic cats by Amblyomma americanum. Parasitol. Res. 112 (Suppl. 1), 11–20. Roosje, P.J., Whitaker-Menezes, D., Goldschmidt, M.H., Moore, P.F., Willemse, T., Murphy, G.F., 1997. Feline atopic dermatitis. A model for Langerhans cell participation in disease pathogenesis. Am. J. Pathol. 151, 927–932. Sager, H., Davis, W.C., Dobbelaere, D.A., Jungi, T.W., 1997. Macrophageparasite relationship in theileriosis. Reversible phenotypic and functional dedifferentiation of macrophages infected with Theileria annulata. J. Leukoc. Biol. 61, 459–468. Schreeg, M.E., Marr, H.S., Tarigo, J., Cohn, L.A., Levy, M.G., Birkenheuer, A.J., 2013. Pharmacogenomics of Cytauxzoon felis cytochrome b: implications for atovaquone and azithromycin therapy in domestic cats with cytauxzoonosis. J. Clin. Microbiol. 51, 3066–3069. Sica, A., Mantovani, A., 2012. Macrophage plasticity and polarization: in vivo veritas. J. Clin. Invest. 122, 787–795. Snider, T.A., Confer, A.W., Payton, M.E., 2010. Pulmonary histopathology of Cytauxzoon felis infections in the cat. Vet. Pathol. 47, 698–702. Snyder, P.W., 2012. Diseases of immunity. In: Zachary, J.F., McGavin, M.D. (Eds.), Pathologic Basis of Veterinary Disease. , 5th ed. Elsevier, St. Louis, MO, pp. 242–288. Tarigo, J.L., Scholl, E.H., Mc, K.B.D., Brown, C.C., Cohn, L.A., Dean, G.A., Levy, M.G., Doolan, D.L., Trieu, A., Nordone, S.K., Felgner, P.L., Vigil, A., Birkenheuer, A.J., 2013. A novel candidate vaccine for cytauxzoonosis inferred from comparative apicomplexan genomics. PLoS ONE 8, e71233. Tsushima, K., King, L.S., Aggarwal, N.R., De Gorordo, A., D’Alessio, F.R., Kubo, K., 2009. Acute lung injury review. Intern. Med. 48, 621–630. Wagner, J.E., Ferris, D.H., Kier, A.B., Wightman, S.R., Maring, E., Morehouse, L.G., Hansen, R.D., 1980. Experimentally induced cytauxzoonosis-like disease in domestic cats. Vet. Parasitol. 6, 305–311. Wilkins, P.A., Otto, C.M., Baumgardner, J.E., Dunkel, B., Bedenice, D., Paradis, M.R., Staffieri, F., Syring, R.S., Slack, J., Grasso, S., Pranzo, G., 2007. Acute lung injury and acute respiratory distress syndromes in veterinary medicine: consensus definitions: the Dorothy Russell Havemeyer Working Group on ALI and ARDS in veterinary medicine. J. Vet. Emerg. Crit. Care 17, 333–339.
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Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm
Research paper
Local pulmonary immune responses in domestic cats naturally infected with Cytauxzoon felis Karelma Frontera-Acevedo a , Kaori Sakamoto b,∗ a School of Veterinary Medicine, Faculty of Medical Sciences, University of the West Indies, Building 47, Eric Williams Medical Sciences Complex, Uriah Butler Highway, Champ Fleurs, Trinidad and Tobago b Department of Pathology, The University of Georgia, College of Veterinary Medicine, 501 D.W. Brooks Dr., Athens, GA 30602, United States
a r t i c l e
i n f o
Article history: Received 2 July 2014 Received in revised form 10 October 2014 Accepted 29 October 2014 Keywords: Cat Cytauxzoon felis Immune response Lung Macrophage Pathology
a b s t r a c t Cytauxzoonosis is a hemoprotozoal disease of cats and wild felids in the South and Southeastern United States caused by Cytauxzoon felis. Although the causative agent has been recognized since the seventies, no study has examined the local immune response in affected organs, such as the lung, and compared them to the lungs of uninfected domestic cats. Previous studies have suggested that the histopathologic findings in the lungs of C. felis-infected cats are caused by the release of pro-inflammatory mediators, such as cytokines and increased production of inducible nitric oxide synthase (iNOS), by the infected macrophages. Our laboratory had previously found an upregulation of the adhesion molecule CD18, which can stimulate the release of these pro-inflammatory mediators. The objective of this study was to characterize local pulmonary immune responses in cats naturally infected with C. felis. Immunohistochemistry was performed to detect tumor necrosis factor-␣ (TNF-␣), interleukin (IL)-1, IL-6, iNOS, and major histocompatibility complex (MHC) II in 19 lungs from affected cats that died between 2005 and 2013. Results showed increased expression of all of these molecules when compared to lungs from uninfected, healthy cats. Furthermore, MHC II is expressed in the endothelium of C. felis naturally infected cats. These results support that there is a marked, local, pro-inflammatory immune response that can contribute to the pathogenesis of cytauxzoonosis in the lungs. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Cytauxzoonosis is a fatal disease of domestic cats in the Midwestern, Mid-Atlantic, Southeastern, and Southcentral United States, caused by Cytauxzoon felis, a tick-borne parasite in the order Piroplasmida, family Theileriidae. An infected tick transmits C. felis while feeding, followed by schizogeny of the parasite in monocytes/macrophages
Abbreviation: iNOS, inducible nitric oxide synthase. ∗ Corresponding author. Tel.: +17065425844; fax: +706 542 5828. E-mail address: [email protected] (K. Sakamoto). http://dx.doi.org/10.1016/j.vetimm.2014.10.012 0165-2427/© 2014 Elsevier B.V. All rights reserved.
throughout the body (Fry and McGavin, 2012). While this infection can be asymptomatic, clinical signs in infected cats include anorexia, depression, lethargy, dehydration, pyrexia, dyspnea, icterus, dark urine, and less commonly, pallor, anemic heart murmur, and increased capillary refill time. Hematologic findings may include normocytic, normochromic, nonregenerative anemia, with pancytopenia or moderate neutrophilia (Greene et al., 2006; Reichard et al., 2009). Since its discovery and description in the 1970s, very little has been published regarding the feline immune response to this disease. A previous study reported the formation of antibodies against the non-pathogenic,
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erythrocytic stage of C. felis (Cowell et al., 1988). Kier et al. (1987) confirmed the monocyte/macrophage identity of the infected cells in the leukocytic stage of the disease. Snider et al. (2010) described and categorized the interstitial pneumonia commonly present in cats that died from C. felis infection and suggested that this inflammation is likely caused by release of pro-inflammatory cytokines and chemokines by the infected macrophages. One of the main histopathologic characteristics of cytauxzoonosis is the presence of giant, infected, intravascular monocyte/macrophages, many of which are adhered to the vascular endothelium, with activation and involvement of CD18. Our laboratory had previously found an upregulation of this molecule (Frontera-Acevedo et al., 2013), which can stimulate the release of some pro-inflammatory mediators, such as TNF-␣, IL-1, IL-6, and inducible nitric oxide synthase (iNOS). That paper characterized the systemic immune response to C. felis infection, and supported the hypothesis that cytauxzoonosis causes a robust systemic pro-inflammatory response characterized by increases in pro-inflammatory cytokines, an acute-phase response, IgM deposition on erythrocytes, and upregulation and expression of CD18 on leukocytes. This response is more severe in cats that died of C. felis infection, compared with the response in cats that survived the disease, which suggests that the immune response is important in the pathogenesis of this disease. Immunohistochemical characterization of the local pulmonary immune response can demonstrate the effects of the immune response in one of the main tissues in which pathologic changes due to C. felis infection have been recognized, and which contributes to the high morbidity and mortality of this disease. 2. Materials and methods 2.1. Case selection Nineteen samples of lung tissues from cats that died of cytauxzoonosis and were received for necropsy at the Athens Diagnostic Laboratory between the years 2005 and 2013 were selected for further immunohistochemical studies. For all of the cases selected, the original hematoxylin and eosin slides containing lung tissue were examined by a board-certified, veterinary pathologist. For negative controls, one archived research case from a healthy control cat used in another experiment was used. For comparison purposes, two cats that died of conditions unrelated to cytauxzoonosis or pulmonary disease (hemolytic anemia and cholangiohepatitis) were also examined. 2.2. TNF-˛, IL-1ˇ, IL-6, iNOS, and MHC II immunohistochemistry
CA), mouse anti-feline IL-6 monoclonal antibody (Clone 341031, RnD Systems, Minneapolis, MN), goat anti-feline IL-1 polyclonal antibody (Catalog Number AF1796, RnD Systems, Minneapolis, MN), mouse anti-human HLA-DR, ␣-chain monoclonal antibody (Clone TAL 1B5, Santa Cruz Biotechnology, Santa Cruz, CA), and rabbit anti-mouse iNOS polyclonal antibody (Catalog Number PA3-030A, Thermo Scientific, Rockford, IL), and their appropriate isotype control antibodies (RnD Systems, Minneapolis, MN, AbD Serotec, Raleigh, NC). The TNF-␣, HLA-DR, and iNOS antibodies have been found to cross-react in cats (Fernandez et al., 2008; Islam et al., 2012; Pullen et al., 1997; Roosje et al., 1997). Two different kits were used for stain development (Dako, Carpinteria, CA; RnD Systems, Minneapolis, MN), based on published protocols and manufacturer’s recommendations. Each slide was incubated with primary antibody either overnight at 4 ◦ C (TNF-␣, IL-1, IL-6, iNOS), or at room temperature for 3 h (MHC II). Slides were counterstained with Gill’s hematoxylin stain and rinsed with deionized water, and a coverslip was then added. More details about these procedures are presented in Table 1. Staining distribution was subjectively compared via light microscopy by a board-certified veterinary pathologist. Immunostained slides were examined in one session per molecule to reduce variability, and a repeat analysis was performed to confirm reproducibility prior to statistical analysis. The TNF-␣ immunoreactivity was characterized as low, moderate, or high. For TNF-␣, low staining was defined as extremely light, diffuse, background staining (but more staining than for negative control samples) and no cytoplasmic staining in the inflammatory cells. Moderate staining was defined as diffuse TNF-␣ immunoreactivity evident in the cytoplasm of less than half of the inflammatory (mostly infected) cells. High staining was defined as staining evident in more than half of the inflammatory cells. The IL-1, IL-6, and iNOS immunoreactivity was characterized as low or high. For both cytokines and iNOS, low staining was defined as extremely light, diffuse, background staining (but more staining than for negative control samples) and scattered intracytoplasmic staining in few scattered alveolar macrophages. High staining was characterized as more diffuse intracytoplasmic staining present in at least half of the various cell types examined. The MHC II immunoreactivity was characterized as low, moderate, or high. For MHC II, low staining was defined as staining of only the cell membrane of a few alveolar macrophages. Moderate staining was defined as either staining of cells besides alveolar macrophages or plasmalemmal staining of the endothelium, but not both present at the same time. High staining was defined as staining of both leukocytes and the endothelium. 2.3. Statistical analysis
Tissues were fixed in 10% neutral-buffered formalin, embedded in paraffin, cut at a thickness of 4 m, and placed on glass slides. If antigen retrieval was performed, a 10 mM sodium citrate solution (pH 6.0) was used. Antibodies used included: goat anti-mouse TNF-␣ polyclonal antibody (Clone M-18, Santa Cruz Biotechnology, Santa Cruz,
For immunohistochemical analysis, a Fischer’s exact test (InStat, Graphpad Software, La Jolla, CA) was performed to determine significant differences between infected and uninfected groups in terms of expression of TNF-␣, IL-1, IL-6, iNOS, and MHC II.
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Table 1 Primary antibodies, dilutions, and antigen retrieval methods used. N/A = no antigen retrieval used, ABC = avidin–biotin complex kit, Polymer = polymer/streptavidin kit. Primary antibody
Dilution
Final concentration (g/mL)
Antigen retrieval method
Detection method
TNF-␣ IL-1 IL-6 iNOS MHCII
1:100 1:10 1:10 1:100 1:50
N/A 10 50 N/A 4
L.A.B Citrate N/A Citrate Citrate
Polymer ABC ABC ABC Polymer
3. Results 3.1. Histopathology results The major pulmonary histopathologic findings are summarized in Table 2. Of the nineteen cases examined, four lungs were diagnosed by the anatomic pathologist who performed the necropsy as pneumonia (three interstitial pneumonias, one bronchopneumonia), and three were diagnosed with vasculitis, not just limited to the lung lesions. Fig. 1 exemplifies all of the major histopathologic findings present. 3.2. Pro-inflammatory cytokines and iNOS immunohistochemistry All lung tissues obtained from cats that died of C. felis stained positively for TNF-␣ by immunohistochemistry. The tissues were characterized as having high staining, particularly in the cytoplasm of infected macrophages that lined vessels and other inflammatory cells within the pulmonary interstitium and alveoli (Fig. 2A). Of the 19 lung tissues from cats that died of C felis, 16 had moderate staining for IL-1 by immunohistochemistry, particularly in the cytoplasm of infected cells that lined vessels and other inflammatory cells within the pulmonary interstitium and alveoli (Fig. 3A). Occasional staining was also noted in the cytoplasm of endothelial cells and surrounding alveolar macrophages. The lung tissues from the three uninfected cats had low staining for IL-1, and in
some cases, complete absence of staining (Fig. 3C). A significant difference (P = 0.013) in staining for IL-1 was observed between cats that died of cytauxzoonosis and the uninfected cats. Of the 19 lung tissues from infected cats, 16 had moderate staining for IL-6 by immunohistochemistry, particularly in the cytoplasm of infected cells and uninfected monocytes, alveolar macrophages, lymphocytes, and neutrophils, with rare intracytoplasmic staining of the vascular endothelium (Fig. 4A). Only one case from an infected cat had low immunoreactivity for both IL-1 and IL-6; another two cases that had low immunoreactivity for IL-6 did had high immunoreactivity for IL-1. The lung tissues from the three uninfected cats also had low or absent staining for IL6 (Fig. 4C). A statistically significant difference (P = 0.013) in staining for IL-6 was observed between cats that died of cytauxzoonosis and the uninfected cats.
3.3. iNOS immunohistochemistry Fourteen lung tissues from infected cats had moderate immunoreactivity for iNOS, while only five lung tissues from infected cats had low immunoreactivity. All tissues from uninfected cats had low immunoreactivity to iNOS. The staining was characterized by cytoplasmic staining in the infected cells, as well as in the surrounding monocytes and alveolar macrophages (Fig. 5). One case that had low staining for IL-6 also had low staining for iNOS, but the other four cases that had low staining for iNOS had moderate staining for both IL-1 and IL-6. A significant difference (P = 0.036) in staining for iNOS was observed between C. felis-infected cats and uninfected cats.
3.4. MHC II immunohistochemistry
Fig. 1. Typical histopathologic appearance of C. felis-infected lung. Note the enlarged, intravascular, infected monocytes, interstitial pneumonia, and alveolar edema.
Immunohistochemistry for MHC II revealed that expression was extremely low in all uninfected cats (Fig. 6C) and was restricted to cytoplasmic staining of a few scattered interstitial macrophages. Cats that died of C. felis infection had moderate (3) to high (16) MHC II expression, as indicated by cytoplasmic staining for MHC II in various cells (Fig. 6A), including in the endothelium. Only three lung tissues from cats that died of cytauxzoonosis did not demonstrate staining of infected cells, but had MHC II cytoplasmic immunoreactivity in the endothelium. A significant difference (P = 0.0006) in staining for MHC II was observed between the cats that died of the disease and uninfected cats.
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Fig. 2. Immunohistochemistry for TNF-␣. (A) Section of lung from a C. felis-infected cat, using anti-TNF-␣ antibody. Note high intensity of immunoreactivity, particularly within the cytoplasm of infected, intravascular monocytes and interstitial leukocytes. (B) Section of lung from a C. felis-infected cat, using isotype antibody. Note background staining of leukocytes, most likely due to endogenous peroxidases. (C) Section of lung from an uninfected cat, using anti-TNF-␣ antibody. Note occasional immunoreactive interstitial leukocytes.
Fig. 3. Immunohistochemistry for IL-1. (A) Section of lung from a C. felis-infected cat, using anti-IL-1 antibody. Note high intensity of immunoreactivity, particularly within the cytoplasm of infected, intravascular monocytes and interstitial leukocytes. (B) Section of lung from a C. felis-infected cat, using isotype antibody. Note slight background staining of infected, intravascular monocytes. (C) Section of lung from an uninfected cat, using anti-IL-1 antibody. Note absence of immunoreactivity.
Fig. 4. Immunohistochemistry for IL-6. (A) Section of lung from a C. felis-infected cat, using anti-IL-6 antibody. Note high intensity of immunoreactivity, particularly within the cytoplasm of infected, intravascular monocytes. (B) Section of lung from a C. felis-infected cat, using isotype antibody. Note slight background staining of infected, intravascular monocytes and interstitial leukocytes. (C) Section of lung from an uninfected cat, using anti-IL-1 antibody. Note absence of immunoreactivity.
Fig. 5. Immunohistochemistry for iNOS. (A) Section of lung from a C. felis-infected cat, using anti-iNOS antibody. Note high intensity of immunoreactivity, particularly within the cytoplasm of infected, intravascular monocytes. (B) Section of lung from a C. felis-infected cat, using isotype antibody. Note absence of immunoreactivity. (C) Section of lung from an uninfected cat, using anti-iNOS antibody. Note absence of immunoreactivity.
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Table 2 Distribution of major pulmonary histopathologic findings in C. felis-infected cats. Numbers are out of a total of 19 C. felis-infected cases that were selected for immunohistochemistry. Major pulmonary histopathologic findings Edema
Alveolar hemorrhage
Thickened septa
Neutrophils within septa
Alveolar macrophages
19
12
11
9
16
4. Discussion Although a complete detailed analysis of histopathologic findings was not performed as described by Snider et al. (2010), a combination of the main significant findings he observed was present in all of our cases. The pulmonary lesions in six of the cases studied were originally reported as interstitial pneumonia or vasculitis. Characteristics of interstitial pneumonia included edema, thickening of the interstitium by edema and neutrophilic infiltrate, and alveolar neutrophilic exudate, among others (López, 2012). At least two of these lesions were present in all lung sections examined, supporting that infection with C. felis causes an interstitial pneumonia. There were also three cases that were interpreted to have vasculitis, an inflammatory lesion that can be a component of systemic disease (Snyder, 2012), such as C. felis infection. Characteristics of vasculitis include the presence of leukocytes within and around the affected vessels, as well as damage to the vessel walls, usually characterized by necrosis or fibrin deposits (Miller et al., 2012). In the cases examined, although necrosis was not noted and fibrin was rare, the common presence of alveolar hemorrhage implies that there had been damage to the vessel walls. In addition, in many cases, neutrophils were observed surrounding the small- to medium-caliber vessels and the alveolar capillaries. Immunohistochemistry of C. felis – infected tissue samples revealed a significant, widespread, qualitative increase in the expression of the pro-inflammatory cytokines TNF␣, IL-1, and IL-6, as well as iNOS, compared with results from uninfected tissues. Detection of these molecules in uninfected control cats was either completely absent or very limited and scattered. A study characterizing the morphologic features of feline infectious peritonitis (FIP) (Kipar et al., 2005) described similar increases in the proinflammatory cytokines TNF-␣ and IL-1 in the tissues of
FIP-infected cats, and related this expression to upregulation by CD18, which has also been suggested by other studies (Kipar et al., 2005; Lee et al., 2000; Leite et al., 2003), as well as our previous study on systemic immune responses to C. felis infection (Frontera-Acevedo et al., 2013). That study found not only a qualitative increase in CD18 expression, but also a relative quantitative increase in CD18 transcription in cats that died of C. felis infection compared to uninfected cats; therefore, the qualitative increase in the pro-inflammatory cytokines observed in this study may be related to the increase in the expression of CD18. The occasional immunoreactivity for pro-inflammatory cytokines noted on the endothelium was also described in the vessels of FIP-infected cats (Kipar et al., 2005), and this was explained in that article as a local paracrine effect of the infected macrophages on the endothelium, instead of a systemic response. Unlike the other two cytokines, IL-6 was found to be less disseminated, perhaps because IL-6, although still part of the innate immune response, is produced slightly later than the other two cytokines (Frontera-Acevedo et al., 2013). Although no previous study has identified the expression of TNF-␣, IL-1, and IL-6 in tissues from C. felis-infected cats, expression of all of these cytokines has been found to be increased in cell lines infected with Theileria annulata (Brown et al., 1995; McGuire et al., 2004). A previous study on C. felis has already addressed the possibility of proinflammatory cytokines being involved in the pathogenesis of the pulmonary lesions (Snider et al., 2010), and the present immunohistochemistry study confirms the extensive presence of TNF-␣, IL-1, and IL-6 in the lung. The strong iNOS immunoreactivity present in the infected cases demonstrates that the infected macrophages are likely actively producing reactive nitrogen intermediates, which is absent in resting macrophages and monocytes, but is induced in response to activation
Fig. 6. Immunohistochemistry for MHC II. (A) Section of lung from a C. felis-infected cat, using anti-MHC II antibody. Note high intensity of immunoreactivity, particularly within the cytoplasm of infected, intravascular monocytes. Inset: High magnification of endothelium from another case showing strong cytoplasmic immunoreactivity of endothelial cells. (B) Section of lung from a C. felis-infected cat, using isotype antibody. Note absence of immunoreactivity. (C) Section of lung from an uninfected cat, using anti-iNOS antibody. Note absence of immunoreactivity.
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(Abbas et al., 2010). This combined expression of proinflammatory cytokines and iNOS in infected macrophages demonstrates that they are activated, as was suggested in a previous study (Snider et al., 2010), and this contributes to the histologic lesions, morbidity, and mortality characteristically observed with this disease. iNOS mRNA expression has been found to be increased in Theileria parva infection (Okagawa et al., 2012), but decreased expression of NO and iNOS has been shown in a study of T. annulata-infected macrophages (Sager et al., 1997). The expression of pro-inflammatory cytokines (TNF-␣, IL-1, IL6) and iNOS is an immunohistochemical feature also found in what is considered acute pulmonary distress syndrome (ARDS), both in humans and animals (López, 2012; Tsushima et al., 2009). Snider et al. (2010) had suggested that cats that died from cytauxzoonosis may be affected with ARDS, based on the histopathologic findings. ARDS is a clinical diagnosis that, in veterinary medicine, has been defined as a condition meeting four of the following five criteria: (1) acute onset of tachypnea and difficulty breathing, (2) presence of risk factors (which includes inflammation, sepsis, or infection), (3) evidence of pulmonary capillary leakage without increased pulmonary capillary hydrostatic pressure, (4) inefficient gas exchange, and (5) evidence of diffuse pulmonary inflammation (López, 2012; Wilkins et al., 2007). One of the main features of ARDS is the increase in alveolar septal and vascular permeability, something that was noted in both Snider’s et al. (2010) study and the present study. One histopathologic feature, though, that is commonly found in human cases of ARDS, but was rarely found by either Snider or this study, is the presence of hyaline membranes lining the alveolar septa (Matthay and Zemans, 2011). Although ARDS is usually caused by sepsis, it is also on occasion caused by a parasitic infection, such as malaria in humans, which is caused by various Plasmodium spp. (Autino et al., 2012; Bauer et al., 2006). In those cases, parasite sequestration in the vascular endothelium causes release of pro-inflammatory mediators (TNF-␣, IL-1, IL-6, NO) and resultant tissue damage. The immunohistochemical surface detection of MHC II was also increased in C. felis-infected lung samples compared to uninfected lungs. This is not only due to the marked increase of antigen-presenting cells in the infected tissue, but also in the expression of MHC II by the endothelium. Endothelial cells are not cell types associated with constitutive MHC II expression, but MHC II expression can be activated and induced by immune mediators, such as IFN-␥ or TNF-␣ in various species (Abbas et al., 2010; Batten et al., 1996; Kariuki Njenga and Dangler, 1995). We recently showed a marked increase in TNF-␣ concentrations serologically in C. felis-infected cats (Frontera-Acevedo et al., 2013), and it is possible that this cytokine was responsible for the induction of MHC II expression in endothelial cells. Unlike endothelial expression of pro-inflammatory cytokines and other molecules, endothelial expression of MHC II happened independently of the presence of nearby infected (and immunoreactive) macrophages. In three cases, the infected macrophages failed to stain for MHC II, but had marked endothelial MHC II immunoreactivity. This endothelial MHC II expression
was considered a systemic response in a study of FIPinfected cats (Kipar et al., 2005), unlike the endothelial expression of pro-inflammatory cytokines. Activated endothelium can also modulate inflammatory responses and can contract (Ackermann, 2012), allowing fluid to leak into the extravascular space and resulting in the edema that was noted in the lungs histologically in this and other studies (Snider et al., 2010). They can also stimulate production and secretion of NO, which although usually a vasodilator, can in large amounts increase inflammation and damage the surrounding tissues (Abbas et al., 2010; Ackermann, 2012). As noted by immunohistochemistry, the endothelial cells in this study did occasionally express iNOS, and the overexpression of NO by vascular walls is known to contribute to certain shock symptoms, such as hypotension and microvascular damage (Forstermann and Sessa, 2012). These effects can then cause congestion and edema, which are part of the common pathologic findings noted in C. felis-infected cats (Greene et al., 2006). A limitation of these immunohistochemical studies is that, while they helped to characterize the local immune response and confirm the presence of pro-inflammatory cytokines in the affected tissues, they cannot measure the local concentration of cytokines produced, or the relative increase in cytokine mRNA expression. In our previous study (2013), we were able to demonstrate a marked increase in the serum concentrations of TNF-␣ and IL-1 in cats that had died of the disease compared to cats that survived, but it was limited in that the disease course was not uniform because the sample population consisted of naturally infected cats. The early studies that characterized cytauxzoonosis in cats by experimental infection described the gross and histologic characteristics of the disease (Kier et al., 1982a,b; Wagner et al., 1980), but at that time did not elucidate immunopathologic mechanisms. The lack of a C. felis in vitro culture system also limits the study of this disease without the infection of live cats.
5. Conclusions Overall, the findings in this paper confirm previous suggestions (Frontera-Acevedo et al., 2013; Snider et al., 2010) that the C. felis-infected macrophages are activated in a classical or M1 fashion, secreting and/or expressing various pro-inflammatory mediators. Although, typically, a M2 macrophage response is expected during parasite infections (Sica and Mantovani, 2012), it is possible that C. felis can somehow alter and modulate the response in the macrophage to ensure survival and replication. The resulting pro-inflammatory local and systemic immune responses are then responsible for the morbidity and mortality associated with this disease. Several articles have been published recently describing various methods of potentially preventing and treating cytauxzoonosis in cats (Lewis et al., 2014; Reichard et al., 2013; Schreeg et al., 2013; Tarigo et al., 2013), but none of them have studied the pathogenesis of the disease or the role of the feline immune system. The findings in this article can be useful in directing the development of new, improved treatments for cytauxzoonosis.
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