Research topic A

Acute pancreatitis is the most common non-malignant gastrointestinal disorder leading to hospitalization. While the majority of patients show a mild, self-limiting course of the disease, in about 20% of cases a severe necrotizing form of pancreatitis develops, accompanied by severe complications and associated with high morbidity and mortality. Severe acute pancreatitis (SAP) is a protease-driven disease accompanied by a systemic immune response. This sterile inflammation is initiated by infiltrating cells of the innate immune system, while the adaptive immune system plays a decisive role in the further disease course, its severity and chronification. Infiltrating macrophages, activated by DAMPs, regulate the systemic immune response by releasing cytokines of the IL-1 family (IL-1β, IL-1α, IL-18 and IL-33), while IL-12 and IL-23 secretion is suppressed. The systemic T cell response is skewed towards a Th2/Treg pattern leading to M2 polarization. The regulation of the immune response is not well understood but plays a crucial role for the disease severity. SAP is still a life-threating disease and, till today, there is no causal treatment available.

We propose that within the damaged pancreas, the protease activity determines activation and degradation of the IL1-family cytokines and thus defines the immune response. The relation between the two relevant concepts of pancreatitis, protease activation and immune response, will be investigated.

Aim 1 | To answer the question whether pancreatic proteases control inflammation by the activation/degradation of cytokines.

Aim 2 | To elucidate the role of cytokines of the IL-1 cytokine family in acute and chronic pancreatitis using suitable knock-out mice of the cytokines IL-1α (C57BL/6J-Il1aem2Lutzy/Mmjax) and IL-33 (C57BL/6-Il33Gt(IST10946B6)Tigm/J) [60].

1. The cytokines of the IL-1 family regulate inflammation during acute and chronic pancreatitis.

Protection from Staphylococcus aureus (S. aureus) requires toxin-neutralizing antibodies, Th1 and Th17 cells. Most S. aureus antigens induce such immune responses in humans. However, S. aureus is also associated with allergies of the respiratory tract and the skin that are characterized by IgE- and Th2 cell dominance. We assume that such allergic (type 2) immune responses facilitate the immune escape of S. aureus, because they counteract the mechanisms that eliminate the bacteria (type 1 and type 3). In fact, patients with respiratory and skin allergies are very densely colonized with S. aureus. We want to clarify the mechanisms used by S. aureus to induce type 2 responses. Two groups of S. aureus antigens stand out because they elicit a type 2-biased immune response in susceptible individuals, the S. aureus superantigens and the serine protein-like proteins (Spls). The latter comprise six serine proteases, SplA - SplF, encoded on a single operon that is present in around 80% of clinical S. aureus isolates. In vitro studies have revealed that each Spl prefers a different cleavage motif, but the enzymes’ pathophysiological substrates are elusive.

From the observation that S. aureus antigens elicit immune responses of distinct quality in the same individual, we conclude that many bacterial proteins have adjuvant properties. This allows a fresh look at an old problem: What makes an allergen an allergen? Specifically: How do the Spls elicit type 2 immune responses?

Aim 1. To identify Spl substrates and to compare the allergenicity of enzymatically inactive Spl mutants with their WT counterparts.

Aim 2. To determine the allergenic properties of Spls.

Properties of allergenic bacterial proteases of S. aureus.

Asthma is one of the most common chronic inflammatory diseases of the lower respiratory tract in all age groups and is associated with significant morbidity and mortality, thus posing a serious public health challenge. About 90% of asthmatics suffer from allergic asthma, which is driven by inhalant allergens and characterized by high titers of allergen-specific IgE and by Th2 cytokines (IL-4 and IL-13). Many common inhalant allergens trigger the airway epithelium to produce cytokines such as TSLP, IL-25, and IL-33. The alarmin IL-33 is a major mediator of the development and maintenance of allergic asthma and promotes differentiation of naïve T cell into Th2 cells. Many common allergens have protease activity and cleave full-length IL-33 into a 10-30 fold more active form. Growing evidence suggests that serine proteases of various origins (bacterial, fungal etc.) also play an important role in the pathogenesis of allergic respiratory diseases. They cleave protease-activated receptors (PARs) in humans and mice. Asthma patients show an increased expression of PAR2 receptors on respiratory epithelial cells, which correlates with the severity of the disease. In addition, activation of PARs on T cells induces a Th2 response. This project will study the role of PARs in S. aureus-serine proteases-induced allergic asthma.

SplB acts as a potent immunodominant allergen in allergic asthma.

Aim 1. To clarify the role of the adaptive immune system in SplB-induced murine allergic asthma.

Aim 2. To analyze the molecular mechanisms of Spl-mediated activation of host cells in vitro.

Murine allergic asthma induced by S. aureus-serine proteases

The species Staphylococcus aureus can colonize and infect a broad range of hosts, with certain S. aureus lineages being associated with particular host species. This host specificity is mediated by host-specific virulence factors, including extracellular proteases. Extracellular proteases can contribute to S. aureus pathogenicity and host adaptation by destroying host extracellular matrix and by evading or manipulating host immune responses.

Our recent studies have revealed that both wild and laboratory mice are natural hosts of S. aureus. The predominant lineage found in laboratory mice is CC88, while CC49 isolates are common in wild mice. These mouse-adapted strains enabled us to set up for the first time a persistent S. aureus colonization model in mice. Interestingly, they frequently produce a novel serine protease, named Jep.

We hypothesize that Jep plays a fundamental role in S. aureus colonization and infection in murine hosts by mediating immune evasion. We will investigate whether Jep disrupts mucosal barriers and degrades extracellular matrix proteins. Moreover, we will investigate the role of Jep in murine colonization and infection.

The role of the Staphylococcus aureus extracellular serine protease Jep in murine colonization and infection

Streptococcus pneumoniae (pneumococci) colonize as a harmless commensal the upper respiratory tract of human. When transmigrating to the lung pneumococci can breach under certain physiological and immunological conditions the epithelial barrier and cause pneumonia and severe invasive diseases. We and others have shown that colonization is facilitated by direct interactions with host cell receptors or via binding to components of the extracellular matrix (ECM) bound to host cell receptors. Similar, pneumococci engage the ECM and serum proteins to evade the innate immune system. To breach the epithelial barrier pneumococci and other bacteria abuse host-derived extracellular proteases or exploit endogenous enzymes and surface-exposed proteases for tissue remodeling processes. We have shown earlier that pneumococci hijack host-derived proteolytic activities like the serine protease plasmin(ogen) to degrade the extracellular matrix, to colonize and invade host tissues. In contrast, the role of pneumococcal serine proteases containing the typical Asp-Ser- His triad essential for their catalytic activity in colonization, mucus degradation, or epithelial barrier disruption is insufficiently explored.

We hypothesize that pneumococcal serine proteases facilitate colonization, pathogen-host interaction, and barrier disruption and that inhibition of serine protease activities attenuate S. pneumoniae.

The role of pneumococcal serine proteases in colonization and barrier disruption.