TGF-ß/IFN-γ Antagonism in Subversion and Self-Defense of Phase II Coxiella burnetii-Infected Dendritic Cells.

Dendritic cells (DCs) belong to the first line of innate defense and come into early contact with invading pathogens, including the zoonotic bacterium Coxiella burnetii, the causative agent of Q fever. However, the pathogen-host cell interactions in C. burnetii-infected DCs, particularly the role of mechanisms of immune subversion beyond virulent phase I lipopolysaccharide (LPS), as well as the contribution of cellular self-defense strategies, are not understood. Using phase II Coxiella-infected DCs, we show that impairment of DC maturation and MHC I downregulation is caused by autocrine release and action of immunosuppressive transforming growth factor-ß (TGF-ß). Our study demonstrates that IFN-γ reverses TGF-ß impairment of maturation/MHC I presentation in infected DCs and activates bacterial elimination, predominantly by inducing iNOS/NO. Induced NO synthesis strongly affects bacterial growth and infectivity. Moreover, our studies hint that Coxiella-infected DCs might be able to protect themselves from mitotoxic NO by switching from oxidative phosphorylation to glycolysis, thus ensuring survival in self-defense against C. burnetii. Our results provide new insights into DC subversion by Coxiella and the IFN-γ-mediated targeting of C. burnetii during early steps in the innate immune response.

Coxiella burnetii-Infected NK Cells Release Infectious Bacteria by Degranulation.

Natural killer (NK) cells are critically involved in the early immune response against various intracellular pathogens, including Coxiella burnetii and Chlamydia psittaciChlamydia-infected NK cells functionally mature, induce cellular immunity, and protect themselves by killing the bacteria in secreted granules. Here, we report that infected NK cells do not allow intracellular multiday growth of Coxiella, as is usually observed in other host cell types. C. burnetii-infected NK cells display maturation and gamma interferon (IFN-γ) secretion, as well as the release of Coxiella-containing lytic granules. Thus, NK cells possess a potent program to restrain and expel different types of invading bacteria via degranulation. Strikingly, though, in contrast to Chlamydia, expulsed Coxiella organisms largely retain their infectivity and, hence, escape the cell-autonomous self-defense mechanism in NK cells.

Chlamydia psittaci-Infected Dendritic Cells Communicate with NK Cells via Exosomes To Activate Antibacterial Immunity.

Dendritic cells (DCs) and natural killer (NK) cells are critically involved in the early response against various bacterial microbes. Functional activation of infected DCs and NK cell-mediated gamma interferon (IFN-γ) secretion essentially contribute to the protective immunity against Chlamydia How DCs and NK cells cooperate during the antichlamydial response is not fully understood. Therefore, in the present study, we investigated the functional interplay between Chlamydia-infected DCs and NK cells. Our biochemical and cell biological experiments show that Chlamydia psittaci-infected DCs display enhanced exosome release. We find that such extracellular vesicles (referred to as dexosomes) do not contain infectious bacterial material but strongly induce IFN-γ production by NK cells. This directly affects C. psittaci growth in infected target cells. Furthermore, NK cell-released IFN-γ in cooperation with tumor necrosis factor alpha (TNF-α) and/or dexosomes augments apoptosis of both noninfected and infected epithelial cells. Thus, the combined effect of dexosomes and proinflammatory cytokines restricts C. psittaci growth and attenuates bacterial subversion of apoptotic host cell death. In conclusion, this provides new insights into the functional cooperation between DCs, dexosomes, and NK cells in the early steps of antichlamydial defense.

Control of Coxiella burnetii shedding in a dairy goat herd by annual offspring vaccination.

A Coxiella burnetii vaccination program, targeting only doelings, was introduced on a German goat farm to curb bacterial shedding. In 2018, adults were vaccinated with a C. burnetii Phase I vaccine at three-weeks apart following pathogen diagnosis, with a booster administered six months later due to sustained high shedding. From 2018 to 2021, doelings received two vaccine doses without any further boosters. To assess the program's efficacy, vaginal swabs from up to 40 animals per age group were collected during kidding seasons from 2019 to 2022. Bulk tank milk (BTM) samples were gathered monthly from January 2018 to October 2022 to monitor herd-level shedding. Real-time PCR analysis determined genome equivalents in all three sample types. Serum samples were taken before the initial immunization and during the post-kidding season from up to 40 goats per age group annually from 2018 to 2022. Phase-specific ELISAs determined IgG Phase I and Phase II antibodies. Additionally, two serum samples per age group from 2022 were analyzed using a neutralization assay. A few goats continued shedding small quantities during subsequent kidding seasons. Although positive BTM samples decreased, they displayed an undulating trend. Most age groups exhibited robust IgG Phase I responses and lower IgG Phase II levels post immunization. Mean IgG levels remained elevated until the study ended compared to pre-vaccination levels in most age groups. Additionally, neutralizing antibodies were present regardless of IgG response. Overall, double vaccination induced lasting antibody levels, but did not entirely prevent C. burnetii shedding. The resilience of the observed humoral immune activity requires further investigation.

Humoral and cellular immune responses in sheep following administration of different doses of an inactivated phase I vaccine against Coxiella burnetii.

An inactivated Coxiella burnetii Phase I (PhI) vaccine (Coxevac®) is licensed in several European countries for goats and cattle to prevent coxiellosis. The vaccine is also applied to sheep, although detailed information about the ovine immune response and vaccine dose is missing. Eighteen gimmers from a C. burnetii unsuspected flock were randomly divided into three groups of six. Group 1 (Cox1) and 2 (Cox2) were vaccinated twice with 1 ml and 2 ml Coxevac®, respectively, three weeks apart (primary vaccination). The same procedure was applied with Cox3 (2 ml sodium chloride, control group). A third injection (booster) was performed after nine months. Potential side effects were determined by measuring the rectal body temperature and skin thickness at the injection site. Blood samples were collected to detect phase-specific IgM and IgG antibodies and interferon-É£ (IFN-É£) release by immunofluorescence assay and ELISAs, respectively. Moreover, a cell infection neutralization assay determined the appearance of neutralizing sera. Body temperatures increased for one day post vaccination, and the skin swelled only slightly. Regardless of the vaccine volume, immunized sheep reacted first with an IgM and IgG PhII response. Ten weeks after the primary vaccination, IgG PhI antibodies predominated. Boosting eight months after primary vaccination resulted in a robust IgG PhI increase and strong IFN-É£ response. In the vaccinated animals, the neutralizing effect is more widespread after the administration of 1 ml than after the treatment with 2 ml. In summary, differences between 1 and 2 ml Coxevac® are minor, and a vaccine volume of 1 ml seems to be sufficient. A booster after the primary vaccination is apparently necessary to stimulate the cell-mediated immune response in naïve sheep.

Chlamydia trachomatis Cell-to-Cell Spread through Tunneling Nanotubes.

Tunneling nanotubes (TNTs) are transient cellular connections that consist of dynamic membrane protrusions. They play an important role in cell-to-cell communication and mediate the intercellular exchanges of molecules and organelles. TNTs can form between different cell types and may contribute to the spread of pathogens by serving as cytoplasmic corridors. We demonstrate that Chlamydia (C.) trachomatis-infected human embryonic kidney (HEK) 293 cells and other cells form TNT-like structures through which reticulate bodies (RBs) pass into uninfected cells. Observed TNTs have a life span of 1 to 5 h and contain microtubules, which are essential for chlamydial transfer. They can bridge distances of up to 50 µm between connecting neighboring cells. Consistent with the biological role for TNTs, we show that C. trachomatis spread also occurs under conditions in which the extracellular route of chlamydial entry into host cells is blocked. Based on our findings, we propose that TNTs play a critical role in the direct, cell-to-cell transmission of chlamydia. IMPORTANCE Intracellular bacterial pathogens often undergo a life cycle in which they parasitize infected host cells in membranous vacuoles. Two pathways have been described by which chlamydia can exit infected host cells: lytic cell destruction or exit via extrusion formation. Whether direct, cell-to-cell contact may also play a role in the spread of infection is unknown. Tunneling nanotubes (TNTs) interconnect the cytoplasm of adjacent cells to mediate efficient communication and the exchange of material between them. We used Chlamydia trachomatis and immortalized cells to analyze whether TNTs mediate bacterial transmission from an infected donor to uninfected acceptor cells. We show that chlamydia-infected cells build TNTs through which the intracellular reticulate bodies (RBs) of the chlamydia can pass into uninfected neighboring cells. Our study contributes to the understanding of the function of TNTs in the cell-to-cell transmission of intracellular pathogens and provides new insights into the strategies by which chlamydia spreads among multicellular tissues.

A Straightforward Hypoxic Cell Culture Method Suitable for Standard Incubators.

We present a new and straightforward method by which standard cell culture plates can be sealed off from ambient air and be placed under controlled hypoxic cell culture conditions without costly or highly specialized materials. The method was established on a murine cell culture system using the dendritic cell line JAWS II but can be readily adapted to other cell cultures. The procedure was designed to be easy to implement in cell culture laboratories with standard incubators and requires only readily available materials, resources, and consumables, such as six-well plates, degassed culture medium, CoCl2, a vacuum sealer, etc., and no further complicated laboratory equipment. The simple hypoxic cell culture method presented here is technically reliable and experimentally safe. As it can be performed in any standard incubator, it is suitable for use at both low and higher biosafety levels.