The impact of serine protease HtrA in apoptosis, intestinal immune responses and extra-intestinal histopathology during Campylobacter jejuni infection of infant mice

Background Campylobacter jejuni has emerged as a leading cause of bacterial enterocolitis. The serine protease HtrA has been shown to be a pivotal, novel C. jejuni virulence factor involved in cell invasion and transmigration across polarised epithelial cells in vitro. However, the functional relevance of the htrA gene for the interaction of C. jejuni with the host immune system in the infant mouse infection model has not been investigated so far. Results Here we studied the role of C. jejuni htrA during infection of 3-weeks-old infant mice. Immediately after weaning, conventional wild-type mice were perorally infected with the NCTC11168∆htrA mutant (∆htrA) or the parental wild-type strain. Approximately one third of infected infant mice suffered from bloody diarrhea until day 7 post infection (p.i.), whereas colonic histopathological changes were rather moderate but comparable between the two strains. Interestingly, parental, but not ∆htrA mutant infected mice, displayed a multifold increase of apoptotic cells in the colonic mucosa at day 7 p.i., which was paralleled by higher colonic levels of pro-inflammatory cytokines such as TNF-α and IFN-γ and the matrix-degrading enzyme matrixmetalloproteinase-2 (MMP-2). Furthermore, higher numbers of proliferating cells could be observed in the colon of ∆htrA infected mice as compared to the parental wild-type strain. Remarkably, as early as 7 days p.i. infant mice also exhibited inflammatory changes in extra-intestinal compartments such as liver, kidneys and lungs, which were less distinct in kidneys and lungs following ∆htrA versus parental strain infection. However, live C. jejuni bacteria could not be found in these organs, suggesting the induction of systemic effects during intestinal infection. Conclusion Upon C. jejuni ∆htrA strain infection of infant mice, intestinal and extra-intestinal pro-inflammatory immune responses were ameliorated in the infant mouse model system. Future studies will shed further light onto the molecular mechanisms of host-pathogen interactions.


Background
Camplylobacter jejuni displays a major infectious agent of foodborne bacterial enterocolitis of men with increasing prevalence in developed as well as developing countries [1,2]. Severity of campylobacteriosis varies from mild disease to acute symptoms such as abdominal cramps, fever, myalgia, and watery to bloody diarrhea [3]. Patients suffering from acute disease display crypt abscesses, ulcerations and colonic infiltration with pro-inflammatory immune cell populations [4][5][6]. Whereas the vast majority of C. jejuni infections is normally self-limiting in humans, post-infectious sequelae such as Guillain-Barré syndrome, Miller Fisher syndrome, Reiter's syndrome and reactive polyarthritis might arise in rare cases [3,7]. An important prerequisite for C. jejuni causing disease is its ability to adhere and invade intestinal epithelial cells [8]. A plethora of bacterial outer membrane proteins such as JlpA, CadF, FlpA, PEB1 among others has been shown to be involved in adhesion to epithelial cells [9][10][11][12][13], whereas CadF can induce the activation of small Rho GTPases, Rac1 and Cdc42, which exert invasive properties in vitro [13][14][15][16] and in human ex vivo biopsies [17]. We and others have recently shown that the C. jejuni serine protease and chaperone HtrA (high temperature requirement A) displays a novel virulence factor [18][19][20][21]. Whereas HtrA family members were considered in the past to strictly act intracellularly in the bacteria, we recently discovered that HtrA is actively secreted into the extracellular environment where it cleaves cell surface adhesion proteins and tumor-suppressor E-cadherin [21][22][23]. In vitro infection experiments with C. jejuni revealed that secreted HtrA is capable of opening cell-to-cell-junctions in the epithelium by cleaving-off the 90 kDa extracellular domain of Ecadherin [21,22]. Furthermore, htrA gene deletion has been shown to result in defective E-cadherin shedding and compromised transmigration of C. jejuni across polarized epithelial cells in vitro [21].
The studies of molecular mechanisms of pathogenhost-interactions causing C. jejuni induced disease have been hampered by a lack of suitable in vivo models given that the host-specific composition of the microbiota determines the physiological colonization resistance against C. jejuni [24,25]. Whereas conventionally colonized adult (>8-weeks-old) mice expel the pathogen within a few days post infection, gnotobiotic wild-type mice and mice recolonized with human microbiota were readily colonized by C. jejuni [24]. However, classical clinical symptoms of human campylobacteriosis such as bloody diarrhea were missing in these murine infection models [24]. In contrast 3-weeks-old infant mice are highly susceptible to C. jejuni infection and develop self-limiting bloody diarrhea within one week [25][26][27][28][29][30]. After resolving enterocolitis within another 7-10 days, infant mice were asymptomatic long-term C. jejuni carriers exhibiting distinct pro-inflammatory immune responses in intestinal as well as extra-intestinal locations such as liver, lungs, and kidneys characterized by influx of predominantly T (and less distinctly B) lymphocytes after more than 3 months p.i. [25,31]. In the present study, we applied the infant mouse model to investigate the functional relevance of the htrA gene in C. jejuni infection in vivo. Furthermore we studied potential extraintestinal inflammatory sequelae in the early course of C. jejuni induced disease.

Results
Intestinal colonization and clinical symptoms in infant mice following infection with wild-type and htrA mutant C. jejuni Immediately after weaning conventional 3-weeks-old infant mice were perorally infected with approximately 10 9 colony forming units (CFU) of either the C. jejuni knockout mutant NCTC11168::htrA (ΔhtrA) or the parental wild-type (WT) strain each harvested in the stationary phase on two consecutive days (day 0 and 1). Control analyses demonstrated that equal amounts of C. jejuni protein were infected per sample and the HtrA protein is not expressed in the ΔhtrA mutant as expected ( Figure 1A). Seven days post infection (p.i.) less than half of parental and ΔhtrA strain infected mice harboured the pathogen in the large intestine (8.3% and 46.2%, respectively) with relatively low pathogenic loads between 10 3 and 10 7 CFU per g luminal content, whereas in the proximal and distal small intestinal tract either C. jejuni strain was virtually undetectable ( Figure 1B). In addition, approximately one third of mice developed clinical symptoms of C. jejuni induced acute enterocolitis until day 7 p.i., as indicated by 33.3% and 38.5% positive cases of bloody diarrhea in parental and ΔhtrA strain infected mice, respectively ( Figure 2A). We next assessed gradual histopathological changes in the mucosa and lamina propria of hematoxylin and eosin (H&E) stained colonic paraffin sections. Seven days following infection with either strain mice displayed comparable histopathological scores and exhibited rather mild to moderate colonic histopathological changes such as single to mild scattered cell infiltrates within the colonic mucosa and submucosa, mild epithelial hyperplasia and starting loss of goblet cells ( Figure 2B).

C. jejuni HtrA aggravates intestinal apoptosis and immune responses
Given that apoptosis is a commonly used diagnostic marker in the histopathological evaluation and grading of intestinal disease [24] and a key feature of C. jejuni induced ulcerative enterocolitis [25], we next quantitatively assessed apoptotic cells applying in situ immunohistochemical stainings of colonic paraffin sections. Seven days following parental, but not ΔhtrA strain infection,  infant mice displayed more than two fold higher colonic caspase3-positive apoptotic cell numbers as compared to naïve controls (p < 0.05 vs. naïve; p < 0.0001 vs. ΔhtrA strain; Figure 3A). Furthermore, ΔhtrA strain infected mice exhibited more colonic Ki67-positive proliferating cells as compared to parental strain infected and naïve controls (p < 0.0005 and p < 0.001, respectively; Figure 3B) indicative for upregulated regenerative epithelial function during immunopathology. Given that recruitment of immune cells is a hallmark of human campylobacteriosis [4,5] we next quantitatively assessed T cell populations in the large intestines of C. jejuni infected mice. Seven days following ΔhtrA strain infection, infant mice displayed a trend towards lower colonic CD3-positive T lymphocyte numbers as compared to parental strain infected control animals (not significant due to high SD in either group; Figure 3C), whereas Foxp3-positive regulatory T cells (Tregs) increased comparably in the colonic mucosa and lamina propria of mice upon peroral infection with either strain ( Figure 3D).
C. jejuni HtrA is necessary for the induction of TNF-α, IFN-γ and matrixmetalloproteinase-2 We next determined C. jejuni induced pro-inflammatory cytokine responses in colonic ex vivo biopsies. Colonic TNF-α protein and IFN-γ mRNA expression levels increased almost two-fold 7 days following parental, but not ΔhtrA infection (p < 0.01 and p < 0.05, respectively; Figure 4) further underlining the role of HtrA in aggravating C. jeuni mediated inflammation.   Given that the matrix-degrading endopeptidase matrixmetalloproteinase (MMP)-2 is upregulated during intestinal immunopathology in mice and men [32][33][34][35][36], we next assessed colonic expression levels of MMP-2 and its endogenous inhibitor, the tissue inhibitor of matrixmetalloproteinase (TIMP)-1. Seven days following C. jejuni infection, MMP-2 mRNA expression levels increased multi-fold, but less distinctly in colons of ΔhtrA as compared to parental strain infected mice (p < 0.05; Figure 5A). Furthermore, colonic TIMP-1 mRNA expression was upregulated 7 days following parental but not ΔhtrA strain infection (p < 0.01; Figure 5B). Taken together, absence of the htrA gene is associated with less distinct C. jejuni induced apoptosis and inflammation in the intestinal tract.

C. jejuni HtrA plays a crucial role in the induction of extra-intestinal immune responses
We have recently shown that asymptomatic longterm C. jejuni carrying mice displayed inflammatory immune responses in extra-intestinal compartments such as liver, kidneys and lungs [30]. We here investigated extra-intestinal C. jejuni induced sequelae as early as 7 days p.i. To address this we quantified inflammatory changes in H&E stained paraffin sections of liver, kidneys and lungs applying respective standardized histopathological scores. Remarkably, 7 days following C. jejuni infection with either strain mild to moderate histopathological changes could be observed in extraintestinal organs that were exclusively lacking viable C. jejuni ( Figure 6). Whereas only subtle inflammatory infiltrates could be observed in livers of infected infant mice irrespective of the C. jejuni strain ( Figure 6A), histopathological scores for kidneys and lungs were lower in ΔhtrA mutant as compared to parental strain infected animals at day 7 p.i. (Figure 6B, C), indicative for less distinct inflammatory disease in the respective extra-intestinal organs due to htrA deficiency of the C. jejuni strain.
Taken together, upon ΔhtrA strain infection of infant mice large intestinal pro-inflammatory immune responses were ameliorated whereas compensatory regenerative/ proliferating properties of the epithelium were preserved. Remarkably, C. jejuni induced inflammatory sequelae in extra-intestinal organs such as liver, kidneys and lungs could be observed as early as 7 days p.i., whereas extraintestinal responses were less pronounced in the latter two compartments due to htrA deficiency.

Discussion
We have recently shown in vitro that the chaperone and serine protease HtrA secreted by C. jejuni exerts a novel pathogenicity factor that is involved in bacterial invasion and transmigration across epithelial cells by cleaving Ecadherin and opening cell-to-cell junctions [20][21][22][23]. In the in vivo study presented here we investigated the impact of the htrA gene in pathogen-host-interaction and induction of immunopathology upon C. jejuni infection. To address this, conventionally colonized infant mice were infected either with the C. jejuni knockout mutant NCTC11168ΔhtrA or its syngenic parental WT strain at the age of 3 weeks immediatedly after weaning. Even though only a subset of mice harboured the respective strain in the intestinal tract, about one third of infected mice suffered from bloody diarrhea. In a previous infection study with another C. jejuni strain (B2), having highly efficient colonizing properties, virtually all infant mice harboured the pathogen at day 7 p.i., whereas up to 90% of mice displayed bloody diarrhea [30]. However, in our experiments with parental strain NCTC11168, but not ΔhtrA mutant infected infant mice exhibited multi-fold increased numbers of colonic apoptotic cells at day 7 p.i. as compared to naïve controls. Conversely, the number of proliferating cells was significantly increased in ΔhtrA but not parental strain infected mice indicative for up-regulated regenerative properties of intestinal epithelial cells thereby counteracting C. jejuni induced tissue damage. Less pronounced intestinal immunopathology due to the absence of HtrA was further underlined by lower expression levels of colonic pro-inflammatory cytokines such as TNF-α and IFN-γ, which have been shown to be key cytokines mediating C. jejuni induced immunopathology in murine infection models with different clinical severity [24,25]. Interestingly, less distinct intestinal immunopathology was accompanied by lower colonic expression levels of the matrix-degrading enzyme MMP-2 and its endogenous inhibitor TIMP-1 seven days following ΔhtrA as compared to the parental strain infection. These MMP expression data are in good agreement with previous studies demonstrating that MMP-2 is up-regulated in acute and chronic small as well as large intestinal inflammation in mice and men [32,33,[35][36][37][38]. For the first time we have now presented evidence that MMP-2 might also play an important role in mediating C. jejuni-induced disease, which is currently further unravelled in ongoing studies.
Surprisingly, rather mild to moderate histopathological sequelae of C. jejuni infection could be detected as early as one week in extra-intestinal organs such as liver, kidneys and lungs. All organ samples were free of viable C. jejuni as shown by negative cultures. In our previous study, C. jejuni B2 strain infected infant mice exhibited histopathological changes in the respective organs more than 100 days p.i. [30] with inflammatory foci consisting mainly of accumulated CD3-positive T cells [31]. Strikingly, in the present study, extra-intestinal histopathological changes in kidneys and lungs were less distinct one week following ΔhtrA as compared to parental strain infection. Hence, absence of the HtrA protein is not only associated with less pronounced intestinal but also extra-intestinal inflammation.
In humans, only very few cases of pathogen-associated disease manifestations affecting liver, lungs, heart or spleen have been reported in severely immuno-compromized patients with C. jejuni bacteremia [39][40][41]. Fauchere and coworkers showed in isolator-raised germfree mice that C. jejuni was cleared from extra-intestinal compartments such as liver and spleen and the circulation within 24 hours following infection most likely due to non-specific bactericidal factors such as phagocytes and complement [42]. Histopathological changes within extra-intestinal organs, however, were not investigated [42]. In the context with our previous observation that CD3-positive cells accumulate at extra-intestinal locations, it is tempting to speculate that potentially pro-inflammatory immune cell populations might be attracted to the extra-intestinal compartments very early following infection before the subsequent clearing of the pathogen. These immune cells might then further reside in the respective organs and explain the sterile inflammatory responses in extra-intestinal tissue sites observed 7 days p.i. as well as in asymptomatic longterm C. jejuni carriers more than 100 days p.i. [30,31].

Conclusions
Our in vivo study using the infant mouse infection model provides clear evidence for the importance of HtrA as a new virulence factor mediating C. jejuni induced intestinal as well as extra-intestinal immune responses. Thus, we describe here the first known C. jejuni mutant with very high motility [21], but having very low potential to trigger intestinal inflammation and bloody diarrhea as compared to WT bacteria. Future studies will further elucidate the underlying molecular mechanisms of C. jejuni-hostinteractions.

Ethics statement
All animal experiments were conducted according to the European Guidelines for animal welfare (2010/63/EU) with approval of the commission for animal experiments headed by the "Landesamt für Gesundheit und Soziales" (LaGeSo, Berlin, Germany; registration numbers G0123/ 12). Animal welfare was monitored twice daily by assessment of clinical conditions.

Mice and C. jejuni infection
All mice were bred and maintained under specific pathogen-free (SPF) conditions in the facilities of the "Forschungseinrichtung für Experimentelle Medizin" (FEM, Charité -Universitätsmedizin, Berlin, Germany). Immediately after weaning, female 3-weeks-old C57BL/6 mice were infected orally with approximately 10 9 viable CFU of the C. jejuni parental WT strain NCTC11168 or the isogenic mutant strain NCTC11168ΔhtrA lacking the htrA gene [21,22] by gavage in a total volume of 0.3 mL PBS on two consecutive days (day 0 and day 1).

Clinical signs of C. jejuni infection, bloody feces
To assess clinical signs of C. jejuni induced infection, the occurrence of blood in fecal samples was determined applying a standardized score (0 points: no blood; 2 points: microscopic detection of blood by the Guajac method using Haemoccult, Beckman Coulter/PCD, Krefeld, Germany; 4 points: overt blood visible) [25,43].
Quantitative analysis of C. jejuni At time of necropsy (day 7 p.i.) live C. jejuni were detected in luminal samples derived from the duodenum, ileum or colon dissolved in sterile PBS by culture as described earlier [24,31]. In brief, serial dilutions of fecal samples were streaked out on karmali agar (Oxoid, Wesel, Germany) and incubated in a microaerobic atmosphere at 37°C for at least 48 hours. The respective weights of luminal fecal samples were determined by the difference of the sample weights before and after asservation.

Cytokine detection in colonic ex vivo biopsies
Colonic biopsies were cut longitudinally and washed in PBS. Strips of approximately 1 cm 2 colon were placed in 24-flat-bottom well culture plates (Nunc, Wiesbaden, Germany) containing 500 μL serum-free RPMI 1640 medium supplemented with penicillin (100 U/ mL) and streptomycin (100 μg/ mL; PAA Laboratories). After 18 h at 37°C supernatants were tested for TNF-α by ELISA (BD Biosciences).

Real-time PCR analysis
RNA was isolated from colonic tissues using the RNeasy Mini Kit (Qiagen). mRNA was reversed transcribed and analysed in triplicate assays by TaqMan PCR using a sequence detection system (ABI Prism 7700; Applied Biosystems) as described previously [35,49]. For detection of murine IFN-γ, MMP-2 and TIMP-1 assays including double-fluorescent probes in combination with assays for the mouse housekeeping gene hypoxanthine phosphoribosyltransferase (HPRT) were purchased from Applied Biosystems). Expression levels were calculated relative to the HPRT expression.

Antibodies and Western blotting
C. jejuni cell pellets were lysed and proteins were separated by SDS-PAGE [50,51]. Coomassie blue staining was done as described [52]. The polyclonal rabbit α-HtrA antibody was raised against a conserved peptide corresponding to amino acid (aa) residues 288-301: C-QGDTKKAYKNQEGA. The peptide was conjugated to Limulus polyphemus haemocyanin carrier protein, and two rabbits each were immunized by Biogenes GmbH (Berlin, Germany) using standard protocols [53]. The resulting antiserum was affinity-purified and the specificity against the proteins in C. jejuni was confirmed by Western blotting [54,55]. Horseradish peroxidase-conjugated anti-rabbit polyvalent sheep immunoglobulin was used as secondary antibody (DAKO Denmark A/S, DK-2600 Glostrup, Denmark). Blots were developed with ECL Plus Western blot reagents (GE Healthcare, UK limited Amersham Place, UK) as described [56,57].

Statistical analysis
Mean values, medians, and levels of significance were determined using Mann-Whitney-U test. Two-sided probability (P) values ≤ 0.05 were considered significant. All experiments were repeated at least twice.