The impact of serine protease HtrA in apoptosis, intestinal immune responses and extra-intestinal histopathology during Campylobacter jejuni infection of infant mice
- Markus M Heimesaat1Email author,
- André Fischer1,
- Marie Alutis1,
- Ursula Grundmann1,
- Manja Boehm2,
- Nicole Tegtmeyer2,
- Ulf B Göbel1,
- Anja A Kühl3,
- Stefan Bereswill†1 and
- Steffen Backert†2
© Heimesaat et al.; licensee BioMed Central Ltd. 2014
Received: 8 March 2014
Accepted: 19 May 2014
Published: 27 May 2014
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.
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.
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.
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 . Patients suffering from acute disease display crypt abscesses, ulcerations and colonic infiltration with pro-inflammatory immune cell populations [4–6]. 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 . 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 [9–13], whereas CadF can induce the activation of small Rho GTPases, Rac1 and Cdc42, which exert invasive properties in vitro[13–16] and in human ex vivo biopsies . We and others have recently shown that the C. jejuni serine protease and chaperone HtrA (high temperature requirement A) displays a novel virulence factor [18–21]. 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 [21–23]. 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 E-cadherin [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.
The studies of molecular mechanisms of pathogen-host-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. However, classical clinical symptoms of human campylobacteriosis such as bloody diarrhea were missing in these murine infection models . In contrast 3-weeks-old infant mice are highly susceptible to C. jejuni infection and develop self-limiting bloody diarrhea within one week [25–30]. 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 extra-intestinal inflammatory sequelae in the early course of C. jejuni induced disease.
Intestinal colonization and clinical symptoms in infant mice following infection with wild-type and htrA mutant C. jejuni
C. jejuni HtrA aggravates intestinal apoptosis and immune responses
C. jejuni HtrA is necessary for the induction of TNF-α, IFN-γ and matrixmetalloproteinase-2
C. jejuni HtrA plays a crucial role in the induction of extra-intestinal immune responses
Taken together, upon ∆htrA 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 extra-intestinal responses were less pronounced in the latter two compartments due to htrA deficiency.
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 E-cadherin and opening cell-to-cell junctions [20–23]. 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 NCTC11168∆htrA 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 . However, in our experiments with parental strain NCTC11168, but not ∆htrA 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 ∆htrA 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 ∆htrA 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, 35–38]. 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.  with inflammatory foci consisting mainly of accumulated CD3-positive T cells . Strikingly, in the present study, extra-intestinal histopathological changes in kidneys and lungs were less distinct one week following ∆htrA 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 [39–41]. 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 . Histopathological changes within extra-intestinal organs, however, were not investigated . 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 long-term C. jejuni carriers more than 100 days p.i. [30, 31].
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 , 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-host-interactions.
Materials and methods
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 109 viable CFU of the C. jejuni parental WT strain NCTC11168 or the isogenic mutant strain NCTC11168∆htrA 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].
Sampling procedures and histopathology
Mice were sacrificed by isofluran treatment (Abbott, Germany). Tissue samples from liver, kidneys, lungs, and intestinal tract (duodenum, ileum, colon) were removed under sterile conditions. Intestinal samples from each mouse were collected in parallel for histopathological, immunohistochemical, microbiological, and immunological analyses. Immunohistopathological changes were determined in samples derived from colon, liver, kidneys and lungs that were immediately fixed in 5% formalin and embedded in paraffin. Sections (5 μm) were stained with H&E, examined by light microscopy (magnification 100× and 400×) and histopathological changes quantitatively assessed by two independent double-blinded investigators applying respective histopathological scoring systems. In brief:
Colonic histopathology (max. 4 points; according to ): 0: no inflammation; 1: single isolated cell infiltrates within the mucosa, no epithelial hyperplasia; 2: mild scattered to diffuse cell infiltrates within the mucosa and submucosa; mild epithelial hyperplasia; starting loss of goblet cells; 3: cell infiltrates within mucosa, submucosa, and sometimes transmural; epithelial hyperplasia; loss of goblet cells; 4: cell infiltrates within mucosa, submucosa, and transmural; severe inflammation; loss of goblet cells, loss of crypts; ulcerations; severe epithelial hyperplasia.
Hepatic histopathology (max. 9 points; modified Ishak score ): Lobular inflammation: 0: normal; 1: minimal inflammation (few inflammatory infiltrates); 2: mild inflammation (increased inflammatory cells, but less pyknotic necrosis); 3: moderate inflammation (marked increase in inflammatory cells and lots of pyknotic necroses); 4: severe inflammation (necrosis); 5: severe inflammation (plus bridging necroses).
Portal inflammation: 0: normal; 1: mild inflammation (<1/3 of portal tracts); 2: moderate inflammation (ca. 1/2 of portal tracts); 3: severe inflammation (>2/3 of portal tracts); 4: severe inflammation (plus portal inflammation disperse into parenchyma).
Renal histopathology (max. 4 points; according to ):
0: normal glomerulus; 1: focal and mild hypercellularity (normal = 3 per segment); 2: multifocal and moderate hypercellularity with capillary dilatation and mild hyalinosis; 3: diffuse hypercellularity (>50% of the tuft) and capillary aneurysm; 4: extensive sclerosis/crescents, tuft obliteration, collapse.
Pulmonal histopathology (max. 4 points, modified according to ):
0: no inflammation; 1: perivascular cuff of inflammatory cells; 2: mild inflammation, extending throughout <25% of the lung; 3: moderate inflammation covering 25-50% of the lung; 4: severe inflammation involving >50% of the lung.
In situ immunohistochemical analyses of 5 μm thin colonic paraffin sections were performed as described previously [24, 25, 30, 31, 48]. Primary antibodies against cleaved caspase-3 (Asp175, Cell Signaling, USA, 1:200), Ki67 (TEC3, Dako, Denmark, 1:100), CD3 (M-20, Santa Cruz, 1:1000), and Foxp3 (FJK-16 s, eBioscience, 1:100) were used. For each animal the average number of positively stained cells within at least six high power fields (HPF, 0.287 mm2; 400× magnification) was determined microscopically by two independent double-blinded investigators.
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 cm2 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 . 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 . 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].
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.
We thank Michaela Wattrodt, Ursula Rüschendorf, Ines Puschendorf, Alexandra Bittroff-Leben, Silvia Schulze, Gernot Reifenberger, Uwe Lohmann, and the staff of the animal research facility for excellent technical assistance, animal breeding and genotyping of mice. We are grateful to Simone Spieckermann for immunohistochemistry staining of colonic sections.
Financial disclosure, grant support
This work was supported by grants from the German Research Foundation (DFG) to UBG (GO363/12-1, CampyGerm; SFB633, TP A7), SB and AF (SFB633, TP A7), AAK (SFB633, TP Z1), MMH (SFB633, TP B6), MA and UG (SFB633, Immuco), and from the German Federal Ministery of Education and Research (BMBF) to SB (TP1.1). The work of SB, MB and NT is supported through a DFG grant (project B10 of CRC-796).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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