Pavel Dutow

A New Role of the Complement System: C3 Provides Protection in a Mouse Model of Lung Infection with Intracellular Chlamydia psittaci

The complement system modulates the intensity of innate and specific immunity. While it protects against infections by extracellular bacteria its role in infection with obligate intracellular bacteria, such as the avian and human pathogen Chlamydia (C.) psittaci, is still unknown. In the present study, knockout mice lacking C3 and thus all main complement effector functions were intranasally infected with C. psittaci strain DC15. Clinical parameters, lung histology, and cytokine levels were determined. A subset of infections was additionally performed with mice lacking C5 or C5a receptors. Complement activation occurred before symptoms of pneumonia appeared. Mice lacking C3 were ∼100 times more susceptible to the intracellular bacteria compared to wild-type mice, with all C3−/− mice succumbing to infection after day 9. At a low infective dose, C3−/− mice became severely ill after an even longer delay, the kinetics suggesting a so far unknown link of complement to the adaptive, protective immune response against chlamydiae. The lethal phenotype of C3−/− mice is not based on differences in the anti-chlamydial IgG response (which is slightly delayed) as demonstrated by serum transfer experiments. In addition, during the first week of infection, the absence of C3 was associated with partial protection characterized by reduced weight loss, better clinical score and lower bacterial burden, which might be explained by a different mechanism. Lack of complement functions downstream of C5 had little effect. This study demonstrates for the first time a strong and complex influence of complement effector functions, downstream of C3 and upstream of C5, on the outcome of an infection with intracellular bacteria, such as C. psittaci.

Chlamydia psittaci: New insights into genomic diversity, clinical pathology, host–pathogen interaction and anti-bacterial immunity

The distinctive and unique features of the avian and mammalian zoonotic pathogen Chlamydia (C.) psittaci include the fulminant course of clinical disease, the remarkably wide host range and the high proportion of latent infections that are not leading to overt disease. Current knowledge on associated diseases is rather poor, even in comparison to other chlamydial agents. In the present paper, we explain and summarize the major findings of a national research network that focused on the elucidation of host-pathogen interactions in vitro and in animal models of C. psittaci infection, with the objective of improving our understanding of genomics, pathology, pathophysiology, molecular pathogenesis and immunology, and conceiving new approaches to therapy. We discuss new findings on comparative genome analysis, the complexity of pathophysiological interactions and systemic consequences, local immune response, the role of the complement system and antigen presentation pathways in the general context of state-of-the-art knowledge on chlamydial infections in humans and animals and single out relevant research topics to fill remaining knowledge gaps on this important yet somewhat neglected pathogen.

The Complement C3a Receptor Is Critical in Defense against Chlamydia psittaci in Mouse Lung Infection and Required for Antibody and Optimal T Cell Response

BACKGROUND The complement system protects against extracellular pathogens and links innate and adaptive immunity. In this study, we investigated the anaphylatoxin C3a receptor (C3aR) in Chlamydia psittaci lung infection and elucidated C3a-dependent adaptive immune mechanisms. METHODS Survival, body weight, and clinical score were monitored in primary mouse infection and after serum transfer. Bacterial load, histology, cellular distribution, cytokines, antibodies, and lymphocytes were analyzed. RESULTS C3aR(-/-) mice showed prolonged pneumonia with decreased survival, lower weight, and higher clinical score. Compared to wild-type mice bacterial clearance was impaired, and inflammatory parameters were increased. In lung-draining lymph nodes of C3aR(-/-) mice the total number of B cells, CD4(+) T cells, and Chlamydia-specific IFN-γ(+) (CD4(+) or CD8(+)) cells was reduced upon infection, and the mice were incapable of Chlamydia-specific immunoglobulin M or immunoglobulin G production. Performed before infection, transfer of hyperimmune serum prolonged survival of C3aR(-/-) mice. CONCLUSIONS C3a and its receptor are critical for defense against C. psittaci in mouse lung infection. In this model, C3a acts via its receptor as immune modulator. Enhancement of specific B and T cell responses upon infection with an intracellular bacterium were identified as hitherto unknown features of C3a/C3aR. These new functions might be of general immunological importance.

Complement Factor C5 but Not C3 Contributes Significantly to Hydrosalpinx Development in Mice Infected with Chlamydia muridarum

ABSTRACT Hydrosalpinx is a pathological hallmark of tubal infertility associated with chlamydial infection. However, the mechanisms of hydrosalpinx remain unknown. Here, we report that complement factor 5 (C5) contributes significantly to chlamydial induction of hydrosalpinx. Mice lacking C5 (C5−/−) failed to develop any hydrosalpinx, while ∼42% of the corresponding wild-type mice (C5+/+) did so following intravaginal infection with Chlamydia muridarum. Surprisingly, deficiency in C3 (C3−/−), an upstream component of the complement system, did not affect mouse susceptibility to chlamydial induction of hydrosalpinx. Interestingly, C5 activation was induced by chlamydial infection in oviducts of C3−/− mice, explaining why the C3−/− mice remained susceptible to chlamydial induction of hydrosalpinx. Similar levels of live chlamydial organisms were recovered from oviduct tissues of both C5−/− and C5+/+ mice, suggesting that C5 deficiency did not affect C. muridarum ascending infection. Furthermore, C5−/− mice were still more resistant to hydrosalpinx induction than C5+/+ mice, even when live C. muridarum organisms were directly delivered into the upper genital tract, both confirming the role of C5 in promoting hydrosalpinx and indicating that the C5-facilitated hydrosalpinx was not due to enhancement of ascending infection. The C5−/− mice displayed significantly reduced lumenal inflammatory infiltration and cytokine production in oviduct tissue, suggesting that C5 may contribute to chlamydial induction of hydrosalpinx by enhancing inflammatory responses.

Role of Capsule and Suilysin in Mucosal Infection of Complement-Deficient Mice with Streptococcus suis

ABSTRACT Virulent Streptococcus suis serotype 2 strains are invasive extracellular bacteria causing septicemia and meningitis in piglets and humans. One objective of this study was to elucidate the function of complement in innate immune defense against S. suis. Experimental infection of wild-type (WT) and C3−/− mice demonstrated for the first time that the complement system protects naive mice against invasive mucosal S. suis infection. S. suis WT but not an unencapsulated mutant caused mortality associated with meningitis and other pathologies in C3−/− mice. The capsule contributed also substantially to colonization of the upper respiratory tract. Experimental infection of C3−/− mice with a suilysin mutant indicated that suilysin expression facilitated an early disease onset and the pathogenesis of meningitis. Flow cytometric analysis revealed C3 antigen deposition on the surface of ca. 40% of S. suis WT bacteria after opsonization with naive WT mouse serum, although to a significantly lower intensity than on the unencapsulated mutant. Ex vivo multiplication in murine WT and C3−/− blood depended on capsule but not suilysin expression. Interestingly, S. suis invasion of inner organs was also detectable in C5aR−/− mice, suggesting that chemotaxis and activation of immune cells via the anaphylatoxin receptor C5aR is, in addition to opsonization, a further important function of the complement system in defense against mucosal S. suis infection. In conclusion, we unequivocally demonstrate here the importance of complement against mucosal S. suis serotype 2 infection and that the capsule of this pathogen is also involved in escape from complement-independent immunity.

DXD Motif-Dependent and -Independent Effects of the Chlamydia trachomatis Cytotoxin CT166

The Gram-negative, intracellular bacterium Chlamydia trachomatis causes acute and chronic urogenital tract infection, potentially leading to infertility and ectopic pregnancy. The only partially characterized cytotoxin CT166 of serovar D exhibits a DXD motif, which is important for the enzymatic activity of many bacterial and mammalian type A glycosyltransferases, leading to the hypothesis that CT166 possess glycosyltransferase activity. CT166-expressing HeLa cells exhibit actin reorganization, including cell rounding, which has been attributed to the inhibition of the Rho-GTPases Rac/Cdc42. Exploiting the glycosylation-sensitive Ras(27H5) antibody, we here show that CT166 induces an epitope change in Ras, resulting in inhibited ERK and PI3K signaling and delayed cell cycle progression. Consistent with the hypothesis that these effects strictly depend on the DXD motif, CT166 with the mutated DXD motif causes neither Ras-ERK inhibition nor delayed cell cycle progression. In contrast, CT166 with the mutated DXD motif is still capable of inhibiting cell migration, suggesting that CT166 with the mutated DXD motif cannot be regarded as inactive in any case. Taken together, CT166 affects various fundamental cellular processes, strongly suggesting its importance for the intracellular survival of chlamydia.

Severity of Allergic Airway Disease Due to House Dust Mite Allergen Is Not Increased after Clinical Recovery of Lung Infection with Chlamydia pneumoniae in Mice

ABSTRACT Chlamydia pneumoniae is associated with chronic inflammatory lung diseases like bronchial asthma and chronic obstructive pulmonary disease. The existence of a causal link between allergic airway disease and C. pneumoniae is controversial. A mouse model was used to address the question of whether preceding C. pneumoniae lung infection and recovery modifies the outcome of experimental allergic asthma after subsequent sensitization with house dust mite (HDM) allergen. After intranasal infection, BALB/c mice suffered from pneumonia characterized by an increased clinical score, reduction of body weight, histopathology, and a bacterial load in the lungs. After 4 weeks, when infection had almost resolved clinically, HDM allergen sensitization was performed for another 4 weeks. Subsequently, mice were subjected to a methacholine hyperresponsiveness test and sacrificed for further analyses. As expected, after 8 weeks, C. pneumoniae-specific antibodies were detectable only in infected mice and the titer was significantly higher in the C. pneumoniae/HDM allergen-treated group than in the C. pneumoniae/NaCl group. Intriguingly, airway hyperresponsiveness and eosinophilia in bronchoalveolar lavage fluid were significantly lower in the C. pneumoniae/HDM allergen-treated group than in the mock/HDM allergen-treated group. We did observe a relationship between experimental asthma and chlamydial infection. Our results demonstrate an influence of sensitization to HDM allergen on the development of a humoral antibacterial response. However, our model demonstrates no increase in the severity of experimental asthma to HDM allergen as a physiological allergen after clinically resolved severe chlamydial lung infection. Our results rather suggest that allergic airway disease and concomitant cellular changes in mice are decreased following C. pneumoniae lung infection in this setting.

An optimized, fast-to-perform mouse lung infection model with the human pathogen Chlamydia trachomatis for in vivo screening of antibiotics, vaccine candidates and modified host–pathogen interactions

Chlamydia trachomatis causes sexually transmitted diseases with infertility, pelvic inflammatory disease and neonatal pneumonia as complications. The duration of urogenital mouse models with the strict mouse pathogen C. muridarum addressing vaginal shedding, pathological changes of the upper genital tract or infertility is rather long. Moreover, vaginal C. trachomatis application usually does not lead to the complications feared in women. A fast-to-perform mouse model is urgently needed to analyze new antibiotics, vaccine candidates, immune responses (in gene knockout animals) or mutants of C. trachomatis. To complement the valuable urogenital model with a much faster and quantifiable screening method, we established an optimized lung infection model for the human intracellular bacterium C. trachomatis serovar D (and L2) in immunocompetent C57BL/6J mice. We demonstrated its usefulness by sensitive determination of antibiotic effects characterizing advantages and limitations achievable by early or delayed short tetracycline treatment and single-dose azithromycin application. Moreover, we achieved partial acquired protection in reinfection with serovar D indicating usability for vaccine studies, and showed a different course of disease in absence of complement factor C3. Sensitive monitoring parameters were survival rate, body weight, clinical score, bacterial load, histological score, the granulocyte marker myeloperoxidase, IFN-γ, TNF-α, MCP-1 and IL-6.