Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2015 Jan 1.
Published in final edited form as: Mucosal Immunol. 2013 Nov 13;7(4):802–817. doi: 10.1038/mi.2013.97

Contrasting immune responses mediate Campylobacter jejuni induced colitis and autoimmunity

Ankit Malik 1,2, Deepika Sharma 2, Jessica St Charles 1,3, Leslie A Dybas 1, Linda S Mansfield 1,2
PMCID: PMC4112758  NIHMSID: NIHMS531784  PMID: 24220299

Abstract

Campylobacter jejuni is a leading cause of foodborne enteritis that has been linked to the autoimmune neuropathy, Guillain Barré Syndrome(GBS). C57BL/6 IL-10+/+ and congenic IL-10−/− mice serve as C. jejuni colonization and colitis models, respectively, but a mouse model for GBS is lacking. We demonstrate that IL-10-/- mice infected with a C. jejuni colitogenic human isolate had significantly upregulated Type1 and 17 but not Type2 cytokines in the colon coincident with infiltration of phagocytes, T cells and Innate Lymphoid Cells (ILC's). Both ILC and T cells participated in IFN-γ, IL-17 and IL-22 upregulation but in a time- and organ-specific manner. T cells were however necessary for colitis as mice depleted of Thy-1+ cells were protected while neither Rag1-/- nor IL-10R blocked Rag1-/- mice developed colitis after infection. Depleting IFN-γ, IL-17 or both significantly ameliorated colitis and drove colonic responses towards Type2 cytokine and antibody induction. In contrast, C. jejuni GBS patient strains induced mild colitis associated with blunted Type1/17 but enhanced Type2 responses. Moreover, the Type2 but not Type1/17 antibodies cross-reacted with peripheral nerve gangliosides demonstrating autoimmunity.

Keywords: Campylobacter jejuni, Colitis, T cells, Guillain Barré Syndrome (GBS)

Introduction

Campylobacter jejuni is a spiral, gram-negative microaerophilic bacterium that is the second most common cause of gastroenteritis in the United States with over 2.4 million campylobacteriosis cases reported annually and many sporadic cases unreported1. The majority of patients ingesting C. jejuni in raw/undercooked meat and unpasteurized milk develop mild to severe gastroenteritis targeting the colon, which is debilitating but self-limiting within 7 to 10 days2,3. Histopathological manifestations include colonic crypt distortion, crypt abscesses, mucin depletion, edema of the colonic lamina propria (cLP) and significant infiltration of granulocytes and mononuclear cells4. Lesions resolve in most patients, but campylobacteriosis can be life threatening in immune-compromised individuals with systemic spread and multi-organ damage5,6. Furthermore, infection with C. jejuni has been linked with serious autoimmune sequelae such as development or flare-up of Inflammatory Bowel Diseases7, Irritable Bowel Syndrome8, Reiter's Arthritis9 and Guillain Barré Syndrome (GBS)10.

Campylobacter jejuni infection is the most common predisposing factor for developing the peripheral neuropathy GBS with 40% of US cases triggered by this bacterium11,12. Recently, the GBS disease burden was estimated at 3000 to 6000 cases per year13. GBS syndrome consists of at least three different subtypes including acute inflammatory demyelinating polyradiculoneuropthy (AIDP), acute motor axonal neuropathy (AMAN) and acute motor and sensory axonal neuropathy (AMSAN). AMAN and AMSAN are axonal subtypes associated with development of autoantibodies that target gangliosides on peripheral nerves; these autoantibodies are thought to result from molecular mimicry10. Indeed, the lipooligosaccharide (LOS) of C. jejuni isolates from GBS patients with antecedent infections have been shown to mimic gangliosides on peripheral nerves including GM1, GD1a and others10,14,15. When bound to peripheral nerves, these antibodies are expected to block nerve conduction by activation of complement and/or by cellular mechanisms16. At present, plasmapheresis and Intravenous Immunoglobulin (IVIg) treatment are the only known treatments with beneficial effect, but are effective in only 60% of GBS patients17. Little is known about host immunological mechanisms that lead to self-limiting gastrointestinal (GI) disease versus severe enteritis or neurological sequelae.

Our rationale was to utilize inbred mice deficient in IL-10 to study factors mediating the development of C. jejuni-induced enteritis and autoimmune manifestations. Innate and adaptive immune cells, epithelial cells and fibroblasts can produce IL-10. It functions to desensitize APC's, down modulate pro-inflammatory cytokine production and suppress proliferation of mononuclear cells 18,19. Genome wide association analysis studies discovered Single Nucleotide Polymorphisms (SNPs) flanking the il10 gene as the most significant locus outside the MHC locus to associate with Ulcerative Colitis, a form of IBD affecting 8-24/10,000 individuals in the US and Europe. SNPs in IL10 also show a significant association with Crohn's Disease, another form of IBD with a similar incidence20.

We have previously established wild type (IL-10+/+) and IL-10-/- mice of various genetic backgrounds as models of C. jejuni colonization and colitis respectively21,22. While the IL-10+/+ mice of C57BL/6, C3H/HeJ and NOD background were stably colonized with C. jejuni (strain NCTC11168) for 35 days post oral inoculation without any adverse clinical or histopathological effects, the IL-10-/- mice of these three genetic backgrounds developed typhlocolitis (inflammation of cecum and colon)22. Thus, the enteritis model of oral inoculation of IL-10-/- mice with C. jejuni essentially involves combining the most strongly associated pathway for susceptibility to IBD (IL10) with the most common causative bacterium for colitis (Campylobacter jejuni) through the natural route of infection. The histopathological features of colitis in IL-10-/- mice also replicate the histopathological features of C. jejuni associated colitis in humans4,21, including C. jejuni invasion of the colonic epithelium followed by ulceration, necrosis and neutrophilic exudates, infiltration of mononuclear and polymorphonuclear cells into the colonic lamina propria and occasionally the muscularis, and crypt distension with abscesses and edema most prominent in the submucosa. These effects were dose independent as the dose range of 102 – 1010 CFU/mouse produced similar levels of pathology21,23. Furthermore, C57BL/6 IL-10-/- mice inoculated with C. jejuni strains obtained from human GBS patients were colonized, but developed little or no colitis24. Recent studies have revealed the importance of diet25, Pattern Recognition Receptors (TLR 2, 4 and 9)26 and particular signaling molecules (NFκB, mTOR, PI3K-γ)27,28 in C. jejuni colonization and induced pathology in wild type or gnotobiotic IL-10-/- mouse models. However, the role of inflammatory mediators-particularly lymphocytes and their secreted cytokines-has not been established in vivo. We hypothesized that differential cytokine responses mediated by lymphocytes in the colon are responsible for C. jejuni induced colitis, protection from colitis and initiation of autoimmune sequelae in the IL-10-/-murine host. In humans, autoreactive IgG1 is the commonly associated antibody subtype after C. jejuni infection and enhanced IgG1 titers also associate with enhanced severity and a poor long term prognosis for GBS cases29. Because IgG1 isotype classically requires TH2 mediated class switching, we further hypothesized that a C. jejuni specific TH2 response generated by the GBS but not the colitogenic strains will lead to induction of autoreactive IgG1.

Results

C. jejuni induced a mixed Type1 and Type17 cytokine and cellular response in IL-10-/- mice

All mice infected with C. jejuni strain 11168 were colonized, with 40-90% of them exhibiting premature mortality24,25. Furthermore, colon histopathology scores were significantly enhanced compared to the TSB inoculated controls24,25. To assess the type of inflammatory reaction(s) C. jejuni induces to cause colitis, C57BL/6 IL-10-/- (IL-10-/-) mice were orally infected with 109 CFU of C. jejuni 11168 or Trypticase Soy Broth (TSB – sham) and observed for clinical signs of enteritis. Mice were euthanized upon showing a severe enteric disease endpoint or at day 35-post inoculation. Tissues were collected during necropsy and analyzed simultaneously. When colon protein extracts were analyzed, primary/innate cytokines (IL-1β and IL-6), Type1 (IFN-γ) and Type17 (IL-17A and IL-22), but not type 2 (IL-4) cytokines were significantly increased in the infected group (Figure 1A). Similar to the colon, plasma IFN-γ, IL-17A and IL-6 were increased significantly in the infected group (Figure 1C), while other cytokine were not detectable. The numbers of macrophages (F4/80+) and T cells (CD3+) infiltrating the lamina propria as quantified by IHC were significantly increased in the infected group (Figure 1B). These data suggest that C. jejuni infection induces a mixed Type1 and Type17 response in infected IL-10-/- mice. Furthermore, there were consistently high correlations between histopathology score and colonic IFN-γ, IL-1β, IL-6, IL-17A and IL-22 levels (Spearman rank correlation factor, rs=0.7-0.9, Suppl. Table 1).

Figure 1.

Figure 1

Open in a new tab

IL-10-/- mice were inoculated with C. jejuni 11168 or TSB and euthanized at humane end point or 35 days post inoculation. A) Colon homogenate ELISA. IL-12p70, TNF-α and IL-17F were only detected in colons of 20% of the mice and are not shown. B) Colon CD3 and F4/80 IHC, at the time of necropsy, the ileocecocolic junction (junction of ileum, cecum and colon) was fixed in formalin and embedded in paraffin. Its sections were stained for CD3 or F4/80 and the number of positively staining cells in the colonic lamina propria were quantified in 10 random high-powered fields. C) Plasma cytokine ELISA. Other cytokines from Figure 1a were not detectable in majority of mice from infected/control group, and are not shown. D) Plasma C. jejuni specific antibody analysis. Anti C. jejuni IgM and IgA was detectable but did not change significantly and is not shown. Data represented is one of three independent experiments with 8 – 10 mice per group. Bar indicates the median, Mann Whitney U test.

IgG subtype class switching in activated B cells is modulated by Type1, Type2 and Type17 cytokines. It has been long established that IFN-γ induces isotype class switching to IgG2a (or IgG2c, an isoform of IgG2a in C57BL/6 background) and IgG3, while IL-4 primarily induces IgG1 class switching30-32. IL-17 was shown to be necessary for autoreactive IgG2b (but not IgG1 or IgG2a) antibodies in an experimental autoimmune myasthenia gravis model 33. Therefore, antibody responses can inform the nature of C. jejuni specific cytokine responses. Consistent with the upregulation of a Type1 and Type17 but not Type2 response by colon and plasma cytokine analysis, plasma levels of C. jejuni specific IgG2b (published previously24), IgG2c and IgG3 but not IgG1 were significantly enhanced in infected mice (Figure 1D). Therefore, plasma IgG analysis reinforced our findings of a mixed Type1 and Type17 response, and also demonstrated the C. jejuni specificity of the response.

We further analyzed the kinetics of cellular and cytokine changes in a kinetic study wherein mice were sacrificed at days 4, 7 and 11 post C. jejuni inoculation. Day 11 was chosen as the end point to avoid the need for premature euthanasia in the infected mice. Histopathology scores evaluated on hematoxylin and eosin stained sections of the ileocecocolic junctions increased with time, reaching significance at day 7 and 11 (Figure 2A). On day 7, histological changes were moderate exhibiting slight epithelial hyperplasia and diffuse or patchy infiltration of mononuclear and polymorphonuclear cells in the colonic and cecal lamina propria that sometimes extended into the muscularis. On day 11 histological changes were more drastic with marked increase in mononuclear and polymorphonuclear cells in the lamina propria and the muscularis. The epithelium was ulcerated along with crypt dysplasia and abscess formation. Neutrophilic exudates were also apparent in the lumen. The numbers of infiltrating neutrophils (CD11bhiGr-1+) and inflammatory myeloid cells (CD11b+MHCII+Gr-1-) were significantly increased at the earliest time point examined (day 4) and continued to increase until the end of experiment (day 11), as assessed by flow cytometry (Figure 2B). C. jejuni specific IgG2b levels were increased significantly at days 7 and 11 post inoculation, while levels of IgG2c and IgG3 were found to be significantly enhanced on day 11. IgG1 levels (Type2 dependent) were not significantly different at any time point after inoculation (Figure 2C). Therefore, C. jejuni specific IgG responses corroborate Type1 and Type17 cytokine responses. Also, as ascertained by Q-PCR for C. jejuni specific gyrA in fecal DNA (Figure 2D) the degree of colonization did increase with time. However, it cannot be determined at this stage if increase in colonization is a cause of and/or a consequence of inflammation. Remarkably, levels of IFN-γ, IL-17A and IL-22 were increased in draining lymph nodes as early as day 4, and continued to rise until day 11 (Figure 3A). However, in the colon, an increase in IFN-γ production was not significant until day 7 and continued to rise at day 11. IL-17, IL-22, TNF-α and MCP-1 were also highest at day 11 in both organs (Figure 3B). This continuous increase in the levels of pro-inflammatory cytokines from colon and lymph nodes reflects the continuous increase in the number of colon infiltrating neutrophils and inflammatory myeloid cells.

Figure 2.

Figure 2

Open in a new tab

IL-10-/- mice were inoculated with C. jejuni or TSB, and sacrificed at indicated days post inoculation. At the time of necropsy, the ileocecocolic junction (junction of ileum, cecum and colon) was fixed in formalin and embedded in paraffin. A) H&E sections were scored in blinded fashion. B) Colon leukocytes were prepared and analyzed for indicated populations by flow cytometry. Dead/dying cells were excluded on the basis of forward and side scatter. All cells were gated on CD19- gate. C) Mice were bled at time of necropsy, and serum was analyzed for indicated antibody subtypes reactive against C. jejuni antigen. Anti-C. jejuni IgM and IgA was detectable but did not change significantly for any time point and is not shown. D) C. jejuni colonization was measured in the feces at the time of necropsy by Q-PCR. E) Representative photomicrographs from H&E stained proximal colon sections. Data is represented as mean±s.e.m; two independent experiments; 5-8 mice per group per time point. Infected group for each time point was compared with control group pooled for each time points by Kruskal-Wallis test and Dunn's post test.

Figure 3.

Figure 3

Open in a new tab

IL-10-/- mice were inoculated with C. jejuni (open circles) or TSB (filled circles) and euthanized at indicated day post inoculation. A) 5 mm of proximal colon or B) 2.5×106 RBC-depleted cells from MLN were incubated in 0.5ml tissue culture media for 48 hours. Supernatant media was clarified and analyzed for indicated cytokines, as well as IL-1β, IL-4, IL-23 and IL-13, which were not detectable. IL-12p70 was detectable from MLN but not the colon. Data is represented as mean±s.e.m; two independent experiments; 5-8 mice per group per time point. Infected group for each time point was compared with control group pooled for each time points by Kruskal-Wallis test and Dunn's post test.

Both Innate Lymphocytes and T cells contribute to Type1 (IFN-γ) and Type17 (IL-17 and IL-22) cytokine production in an organ, time and cell type specific manner

C. jejuni induced colitis is elicited upon infiltration of mononuclear cells and neutrophils in the colon, both in humans and IL-10-/- mice4,21. These mononuclear cells can belong to myeloid or lymphoid lineages. Lymphoid cells can further be categorized into adaptive lymphocytes—B cells (CD3-CD19+) and T cells (CD3+CD19-), and Rag-independent innate lymphocytes. Mucosal innate lymphocytes can further be divided into NK cells (CD19-NKp46+) and relatively novel Innate Lymphoid Cells (ILCs) (Thy1hiLin-) where Lin has the following markers (CD3-CD19-NKp46-Gr-1-MHC-II-CD11b-). T cells, NK cells and ILC's have been shown to be the major producers of IFN-γ, IL-17 and IL-22 in a number of inflammatory diseases34-36. Therefore in a kinetic study, we evaluated the relative contribution of T cell subsets, NK cells and ILC's to the increase in production of IL-17, IL-22 and IFN-γ in the colon and mesenteric lymph nodes (MLN). In both organs, there were significant increases in the absolute number of T cells (CD3+CD19-) and NK cells at day 7 and/or 11, while ILC's were enhanced at day 4 and 7 but decreased to the level of controls by day 11 (Figure 4A-C and 5 A-C). The maintenance of elevated numbers of T cells and NK cells and basal levels of ILCs was also confirmed in a separate experiment at a later time point of day 21 (not shown). Within the T cell compartment, the proportion of CD4+ TH cells doubled in the colon by day 11, while the proportion of γδ+ T cells was unchanged during the course of the experiment (Figure 4A).

Figure 4.

Figure 4

Open in a new tab

IL-10-/- mice were inoculated with TSB or C. jejuni and sacrificed at indicated day post inoculation. Single cell suspension was prepared from colon. T cells, ILCs and NK cells were analyzed for IFN-γ and IL-17 or IFN-γ and IL-22 production by intracellular cytokine staining and flow cytometry following brief restimulation with PMA and ionomycin in presence of brefeldinA. Double positives were relatively rare and data presented here represents total positive. Dead/dying cells were excluded on the basis of forward and side scatter. Data is represented as mean±s.e.m; two independent experiments with 5 - 8 mice per group per time point. Infected group for each time point was compared with control group pooled for each time points by Kruskal-Wallis test and Dunn's post test. Proportion of IL-17+ or IL-22+ cells from CD3+CD4-cells (which are likely CD8+) was not significantly different at any time point, and is not shown.

Figure 5.

Figure 5

Open in a new tab

IL-10-/- mice were inoculated with TSB or C. jejuni and sacrificed at indicated day post inoculation. Single cell suspension was prepared from MLN. T cells, ILCs and NK cells were analyzed for IFN-γ, IL-17 or IL-22 production by intracellular cytokine staining and flow cytometry following brief restimulation with PMA and ionomycin in presence of brefeldinA. Data is represented as mean±s.e.m; two independent experiments with 5 - 8 mice per group per time point. Infected group for each time point was compared with control group pooled for each time points by Mann Whitney U test. Proportion of IL-17+ or IL-22+ cells from CD3+CD4-cells (which are likely CD8+) was not significantly different at any time point, and is not shown.

The proportion of the IFN-γ+ cells was increased, in TH, TC, γδ T and NK cell compartments on day 11 post infection in both organs (Figure 4A-B and 5A-B). The proportion of IL-17+ ILC's (ILC17) increased as early as day 4 in the MLN (Figure 5C), but decreased to control levels at day 7 while the proportion of IL-17+ TH cells increased on days 7 and 11 (Figure 5A). This pattern of IL-17 production initially from ILCs and subsequently by T cells correlated with the kinetics of infiltration of these cells in the colon and MLN. In contrast, the proportion of ILC17 in the colon did not increase until day 11 (Figure 4C), at which time the total number of these cells had decreased to basal level. This demonstrates a selective organ specific activation of this cell type. Furthermore, increases in IL-22+ TH cells and NK cells were observed at day 7 and/or 11 in both the colon and MLN (Figure 4A-B and 5A-B). Therefore, a time and cell type specific Type1 and Type17 response was observed in the colon and MLNs of C. jejuni challenged IL-10-/- mice.

Thy-1+ lymphocytes are necessary for C. jejuni mediated colitis

Thy-1 (CD90) is a cell surface marker expressed by both innate lymphocytes and T cells and consequently Thy-1 depleting antibody was utilized to deplete these cells in the mouse model. Infected and Thy-1 depleted IL-10-/- mice had significantly lower histopathology scores (Figure 6A) as well as decreased numbers of neutrophils, inflammatory myeloid cells, T cells and DC infiltration of colonic tissues (Figure 6B) as compared to mice that were infected but given control antibody injections (11168+CIgG). In fact, the infected and depleted group (11168+α-Thy-1) had numbers similar to the uninfected group (TSB+α-Thy-1). Out of ten mice in the positive control group (11168+CIgG group), two mice had to be euthanized early (day 9 and 21 p.i.) because they reached the threshold of clinical signs. Their tissues were collected during necropsy and appropriately fixed or frozen and analyzed alongside the other samples after the end of the experiment (Day 25 p.i.). Because T cells are necessary for IgG isotype class switching in B cells, Thy-1 depleted and C. jejuni 11168 infected mice had lower levels of C. jejuni specific IgG2b, IgG2c and IgG3 in the plasma as compared to the 11168+CIgG group (Figure 6C). Furthermore, colonic levels of IFN-γ, IL-17 and IL-22 were decreased to basal levels in Thy1-depleted infected mice reinforcing that Thy1+ cells are the major producers of these cytokines (Figure 6D). The success of depleting Thy-1 positive cells with antibody treatments was confirmed in the blood and colon by flow cytometry (Figure S1B).

Figure 6.

Figure 6

Open in a new tab

IL-10-/- mice were orally inoculated with C. jejuni or TSB and injected with α-Thy-1 or CIgG twice weekly starting 3 days before inoculation, and sacrificed at Day 23-24 post inoculation. At the time of necropsy, the ileocecocolic junction (junction of ileum, cecum and colon) was fixed in formalin and embedded in paraffin. A) H&E sections were scored in blinded fashion. B) Colon leukocytes were prepared and analyzed for indicated populations by flow cytometry. C) Mice were bled at time of necropsy, and plasma was analyzed for indicated antibody subtypes reactive against C. jejuni antigen. D) 5mm of proximal colon was washed and incubated in 0.5ml tissue culture media for 48 hours. Supernatant media was clarified and analyzed for indicated cytokines, as well as other cytokines from Figure.1, which were not significantly different. Bar indicates the median, n=10 mice per group. Kruskal-Wallis test followed by Dunn's post test.

Innate immunity is insufficient to mediate severe C. jejuni induced colitis

To assess if innate immunity is sufficient for C. jejuni mediated colitis, Rag1-/- mice on the C57BL/6 background were challenged with C. jejuni alongside C57BL/6 wt mice that serve as a known positive colonization control. While all infected mice were colonized, neither group developed any clinical signs of colitis, and did not have differences in the histopathology scores (Figure 7A) or the numbers of colonic neutrophils, inflammatory myeloid cells or dendritic cells (not shown) at day 32-35 p.i. as assessed by flow cytometry. To determine if IL-10 was responsible for lack of inflammation in Rag1-/-mice, they were given IL-10R blocking antibody after infection. Even after IL-10R blocking, Rag1-/- mice did not develop any clinical signs of colitis, and had no increase in histopathology scores (Figure 7B) or numbers of phagocytes in the colon (not shown) as compared to TSB+α-IL-10R or 11168+CIgG treated groups. C57BL/6 wt mice did develop C. jejuni specific antibody responses similar in nature to that of IL-10-/- mice with significant increases in the IgG2b, IgG2c and IgG3 isotypes and with no difference in IgG1, IgM or IgA (Figure 7C), demonstrating that the nature of C. jejuni antibody responses are independent of IL-10. Taken together, these data, along with the requirement of Thy-1+ cells to produce colitis, show that T cells are necessary for severe C. jejuni induced colitis. C. jejuni colonization correlated with colitis induction in as much as the mouse genetic background that supports inflammation (IL-10-/-) had higher colonization extent by the end of the experiment than the mouse genotypes/treatments that did not (BL/6wt or Rag1-/- or Rag1-/-IL-10R blocked mice) (Figure 7D).

Figure 7.

Figure 7

Open in a new tab

A) C57BL/6 wt or Rag1-/- mice were orally inoculated with C. jejuni or TSB and sacrificed at Day 32-34 post inoculation. B) Rag1-/- mice were injected with α-IL-10R or CIgG after infection. At the time of necropsy, the ileocecocolic junction (junction of ileum, cecum and colon) was fixed in formalin and embedded in paraffin. H&E sections were scored in blinded fashion. C) Mice were bled at time of necropsy, and plasma was analyzed for indicated antibody subtypes reactive against C. jejuni antigen. D) C. jejuni colonization was measured in the feces at the time of necropsy by Q-PCR. Data is represented as median with 9-10 mice per group. Kruskal-Wallis test followed by Dunn's post test.

Both IFN-γ and IL-17 participate in C. jejuni induced colitis

To account for the relative contribution of IFN-γ and IL-17 in C. jejuni mediated pathology, one or both were depleted during the course of the disease by intra-peritoneal injection of neutralizing antibodies. Results showed that both of these cytokines have a positive role in mediating colitis because depleting either IFN-γ or IL-17 or both led to significant decreases in histopathology scores (Figure 8A). Furthermore, depleting IFN-γ or IL-17 significantly decreased or trended towards decreasing the extent of colonic infiltration of neutrophils, inflammatory myeloid cells and T cells, while depleting both had a significant effect (Figure 8B). The number of colon infiltrating ILC's and the proportion of CD4+ TH and γδ+ T cells were unchanged between all groups (not shown). The number of NK cells, however decreased in the IFN-γ depleted groups, suggesting a positive feedback mechanism (Figure 8B). Notably, depleting IFN-γ and IL-17 pushed the response towards Type2 cytokine and antibody induction. IFN-γ depleted groups had decreased levels of C. jejuni specific plasma IgG2c but increased plasma IgG1 (Figure 8C) along with modestly increased levels of IL-4 and IL-13 in the proximal colon (Figure 8D and not shown). IL-17 depleted groups had decreased IgG2b and increased IgM (Figure 8C). The double depleted group also had increased levels of C. jejuni-specific IgA (Figure 8C). Thus, depleting IFN-γ and IL-17 after infection with the colitogenic 11168 prevented colitis and shifted the immune response towards Type2 cytokines and antibodies.

Figure 8.

Figure 8

Open in a new tab

IL-10-/- mice were orally inoculated with C. jejuni and sacrificed at Day 21-22 post inoculation. IFN-γ, IL-17 or both were neutralized by i.p. injection of neutralizing antibodies twice a week for three weeks, starting on the day of inoculation. A) H&E sections were scored in blinded fashion. B) Colon leukocytes were prepared and analyzed for indicated populations by flow cytometry. C) Mice were bled at time of necropsy, and plasma was analyzed for indicated antibody subtypes reactive against C. jejuni antigen. D) Level of IL-4 was measured in the proximal colon homogenate by ELISA. Data is represented as medians or mean±s.e.m., with 10 mice per group. Kruskal-Wallis test followed by Dunn's post test. Only comparisons with CIgG group are shown.

Infection with GBS associated C jejuni strains induces Type2 immunity that is protective for colitis but leads to autoimmunity

Having observed a Type2 response in 11168 infected IL-10-/- mice after IL-17 and IFN-γ neutralization, we hypothesized that the non-colitogenic GBS strains, 260.94 and HB93-13, also induce a blunted Type1/17 but enhanced Type2 response in the IL-10-/- mouse. Indeed, the GBS strains induced significantly blunted Type1/17 mediators (IFN-γ, IL-17, IL-22, IL-6, T-bet) (Figure 9A and not shown) locally in the colon and systemically in the plasma (not shown) when compared to the colitogenic strains 11168, CF93-6 (shown) and CG8421 (not shown). C. jejuni specific IgGb (published previously24), IgG2c and IgG3 responses by the GBS strains were also trending towards a decrease as compared to the colitogenic strains (Figure 9B). In contrast, the GBS strains significantly enhanced the C. jejuni specific Type2 mediators in the colon (IL-4, IL-13, Gata-3) (Figure 9A) and antibody (IgG1) responses (Figure 9B) in the plasma. Remarkably, Type2 but not Type1/17 autoantibodies were detected in plasma of C. jejuni GBS strain challenged mice. These IgG1 subclass autoantibodies reacted against the peripheral nerve gangliosides, GM1 and GD1a, in an ELISA format (Figure 9C and Figure S2). Molecular mimicry along with cytokine milieu was essential for autoimmunity because only the GBS strains induced autoantibodies. Challenge infections with C. jejuni 11168 even in Type2 inducing conditions, i.e., after depletion of IFN-γ and IL-17 (from Figure 8C), did lead to Type2 antibody induction against the C. jejuni antigen, but these antibodies failed to cross-react with the nerve ganglioside autoantigens (not shown). The GBS strains colonized stably to the same extent as the colitogenic 11168 strain at the early time point of day 4 (from Figure 2D), but had lower fecal colonization at the later time points of day 11 (from Figure 2D) or day 16 (Figure 9D). Therefore, 11168 has enhanced colonization prowess that may be related to its enhanced colitogenic ability. For the GBS strain HB93-13, all stably colonized mice (8 out of 10), but none of the uncolonized mice (2 out of 10) tested positive for autoantibodies against GD1a, suggesting stable colonization as essential for inducing significant autoantibody responses.

Figure 9.

Figure 9

Open in a new tab

IL-10-/- mice were orally inoculated with given C. jejuni strains and sacrificed at Day 35 post inoculation, or earlier if requiring euthanasia. A) Expression of indicated mediators in the colon by real time PCR. B) Antibody subtypes reactive against C. jejuni antigen. C) Peripheral nerve gangliosides autoantigens by ELISA. D) C. jejuni colonization was measured in the feces at the indicated days post inoculation for the GBS strains and the time of necropsy for all strains by Q-PCR. Data is represented as median or mean±s.e.m., with 10 mice per group. Kruskal-Wallis test followed by Dunn's post test. No other comparisons were significant.

The blunted Type1 responses elicited by the GBS strain HB93-13 were also confirmed ex vivo by measuring secreted cytokines from wild type mouse splenocytes challenged with colitogenic 11168 and CF93-6, or GBS associated HB93-13 and 260.94 strains using a gentamicin killing assay. 72 hours post challenge; GBS strains produced significantly less IFN-γ, TNF-α, IL-22 and IL-10 than either of the two colitogenic strains, consistent with the in vivo data (Figure 10A). However, they induced similar IL-12p40 and more IL-6 which points towards differential innate immune activation and/or T cell maturation downstream of TH1/TH17/TH2 pathways. To evaluate IL-12p70 induction by the colitogenic/GBS strains, adherent splenocytes alone were challenged individually (Figure 10B). Consistent with enhanced IFN-γ production by colitogenic strains, higher IL-12p70 was produced by them when compared to the GBS strains. In contrast the GBS strains produced more IL-6 and IL-10. As invasion is likely an important feature of Campylobacter-mediated enteritis, epithelial invasion by gentamicin killing assay was performed using the young adult mouse epithelial cell line (YAMC) (Figure 10C). While the non colitogenic GBS strain 260.94 invaded to a slightly lower extent as the colitogenic 11168, the other GBS strain HB93-13 invaded significantly more. Therefore, different outcomes to infection by colitogenic versus GBS strains cannot be explained by differences in epithelial cell invasion but rather by the Type1/17 or Type2 induction by individual strains.

Figure 10.

Figure 10

Open in a new tab

Whole splenocytes A) or adherent splenocytes B) from naïve C57BL/6 wt mouse were challenged with indicated strains and M.O.I's in antibiotic free R10 media. Gentamicin was added after one hour, and cultures were further incubated for 72 hours for whole or 24 hours for adherent splenocytes, upon which supernatants were clarified and analyzed for indicated cytokines by ELISA. IL-17, IL-4 and IL-23 were not detectable with whole splenocytes while IL-12p70 was only detectable with adherent cell culture. C) Invasion and IL-6 elicitation by the indicated strains was assessed on YAMC cells at M.O.I of 100. Data represents mean±s.e.m. of three wells and analyzed by two-way ANOVA for splenocytes or one way ANOVA for epithelial cells followed by Bonferonni's post test. Only comparisons with respect to 11168 for splenocytes or HB93-13 for epithelial cells are shown. One of three indenpdent replicates is shown.

Discussion

In this study, we show that C. jejuni 11168—a known colitogenic strain—induced a mixed Type1 and 17 cytokine response in the colon and the draining mesenteric lymph nodes of the C57BL/6 IL-10-/- mouse colitis disease model. This is consistent with the observations made by Edwards et al, that demonstrated the induction of Type1 and 17 responses from C. jejuni 11168H challenged human colon explants and peripheral blood derived cells ex vivo, along with a novel role for IL-17 in reducing intracellular survival in intestinal epithelial cells37. In our study, these cytokines orchestrated infiltration of neutrophils, macrophages, T cells, NK cells and ILC's in the colonic mucosa and sub-mucosa that closely resembled lesions seen in patients with Campylobacter-induced enteritis. In our model, ILC's were already increased significantly at day 4, peaked at day 7 and decreased to background level by day 11. Increases in T cells and NK cells did not reach significance until day 7 or 11 respectively. By day 4 post infection, the amount of secreted IFN-γ, IL-17 and IL-22 from the MLN's was already increased 2-3 fold, which indicated innate sources for these cytokines since this response is too early for adaptive immunity to arise. We also found that innate lymphocytes as well as T cells participated in the upregulation of IFN-γ, IL-17 and IL-22, but in a time and organ-specific manner. Mirroring the kinetics of their infiltration in the colon, ILC's upregulated IL-17 as early as day 4 in the lymph nodes that switched to an IFN-γ response by day 7 at which time IFN-γ+ and IL-17+ cells were also increased in the TH and γδ cell compartments. Because only adaptive immunity can be antigen specific, it is tempting to speculate that early upregulation of ILC's followed by their downregulation is a mechanism of ensuring an acute response to infection whilst preventing non-specific inflammatory responses that predispose to autoimmunity. However, in the colon, the cytokine production profile from the ILC's over time is similar to that of T cells, and further studies are needed to determine their functional discrepancy in the two organs. Neutrophil and inflammatory myeloid cell numbers in the colon were enhanced significantly at day 4 and continued to increase until the later time points (e.g. day 11) post infection. This was another reflection of the continuous significant increases in pro-inflammatory factors from the colon and MLN's by a combination of non-specific ILC's and adaptive T cells that maintain and specify the responses.

It should be recognized that although ILCs participated in C. jejuni colitis, the innate immune system was unable to induce full blown colitis. Neither Rag1-/- nor IL-10R blocked Rag1-/- mice developed colitis after C. jejuni inoculation. However, Thy-1+ lymphocytes were necessary for the colitogenic response as significantly decreased pathology was observed in adaptive immune competent IL-10-/- mice that were depleted for Thy-1+ cells. These findings demonstrated that T cells were necessary for C. jejuni to elicit colitis. In contrast, Jobin et al., have very recently shown that germ-free Rag2 IL-10 double KO mice of the 129/SvEv background develop colitis after C. jejuni 11168 inoculation28. The difference in outcomes may be attributable to differences in the host genetics (C57BL/6 v/s 129/SvEv) and/or protective effect of host gut microbiota on degree of colonization and immune stimulation. Jobin et al have also recently shown that depletion of CD4+ cells does not affect the pathology in C. jejuni infected gnotobiotic C57BL/6 IL-10-/- mice27. This observation is complicated by the fact that in addition to immature thymocytes and mature TH cells, CD4 is also expressed by macrophages and therefore CD4 depletion cannot be used as a specific tool for TH cell depletion. Nevertheless it does suggest a pro-inflammatory role of other lymphocytic populations in C. jejuni colitis and, in our model, we indeed observed the increase in numbers of IFN-γ+ and IL-17+ cells in non-TH cell compartments, including γδ and CD4-(CD8+) cells. ILC's can also be further divided into Lymphoid Tissue Inducer cells (Lin-Thy1hiCD4+) and LTi-like cells (Lin-Thy-1hiSca-1+) that have been shown to perform both pro- and antiinflammatory functions38-40. Early IL-22 production from LTi's in the colon of Citrobacter rodentium infected BL/6 IL-10+/+ mice was shown to be protective41 while the production of IFN-γ and IL-17 from LTi-like cells in the colons of Helicobacter hepaticus infected Rag. 129 mice was shown to be pro-inflammatory42. Further studies will be necessary to determine the exact role of these subsets in C. jejuni mediated colon pathology.

We have shown that enhanced IFN-γ and IL-17 cytokine secretion induces lesions in C. jejuni induced colitis. Neutralizing either or both of these cytokines prevented the overt histopathological changes associated with the disease, including the infiltration of neutrophils, inflammatory myeloid cells and T cells into the colon. It was notable that neutralizing these cytokines shifted the immune response towards Type2 cytokines (IL-4 and IL-13) and antibody responses (IgG1), alongside increases in C. jejuni specific IgM. The non-colitogenic GBS strains also induced similarly enhanced Type2 and blunted Type1/17 responses, suggesting that C. jejuni mediated colitis can be explained by the balance between Type1/17 and Type2 responses. This may also suggest the mechanism underlying the development of autoantibodies and autoimmune diseases after treatment with TNF-α blockers in humans43-45. It remains to be seen if blocking the Type2 cytokines in the mouse model could lead to induction of colitis thus diverting the expected autoimmune outcomes after challenge with the non-colitogenic GBS patient strains.

GBS, especially the AMAN form, has most commonly been associated with antecedent C. jejuni infection17. It has also been published that antecedent gastrointestinal symptoms are only observed in 50% of GBS patients that are serologically positive for C. jejuni46,47. This suggests that there is no clear cut association between extent of colitis and GBS in humans and at least half of the human GBS cases mirror our mouse model in that infection with GBS strains leads to autoantibodies without antecedent diarrhea. Furthermore, identical to what has been observed in human GBS patients, autoreactive antibodies exclusively of the IgG1 isotype were found in mice infected with the GBS strains but not the colitogenic strains. Molecular mimicry and not just the cytokine milieu is essential for autoantibody development because the colitogenic non-GBS strain C. jejuni 11168 failed to induce autoantibodies even when the immune response was biased towards Type2 after depleting IFN-γ and IL-17.The non-colitogenic GBS strains were colonized to a similar extent as the colitogenic 11168 strain, but only at the early time point (day 4). Colonization with C. jejuni 11168 was higher at later time points (day 11/16). Therefore 11168 has enhanced colonization prowess that may be related to its enhanced colitogenic ability and Type1/17 over Type2 maturation. However there is still proof of strain-intrinsic but colonization-independent bias of Type1/17 maturation from ex vivo experiments. Stable colonization was nevertheless necessary to develop autoantibodies because 2 of mice infected with the GBS strain HB93-13 strain that were not colonized failed to develop a significant autoantibody level when compared to their 8 colonized group mates. Consistent with our in vivo findings, Bax et al have shown in vitro that LOS purified from GBS strains induced blunted TH1 and enhanced TH2 response when compared to LOS from non-GBS strains48. The TH2 response depended on the nature of sialylation of the LOS and correlated with binding to Sialoadhesin, a DC and macrophage cell surface receptor. The role of Sialoadhesin in C. jejuni phagocytosis and primary interferon induction has also been established49,50. These results suggest that surface modification of the C. jejuni GBS strains play an important role in development of autoimmunity, but more work is needed to determine the relevant ligand-receptor and downstream interactions in this model. Future studies are underway to determine the pathological and histological consequences of developing these autoantibodies, and the innate immune mechanism of differential T cell responses by colitogenic versus GBS strains. It is imperative to understand the host factors and bacterial ligands that are responsible for GBS and IBD in order to develop the needed novel therapeutic interventions for these conditions.

Materials and Methods

Mice

C57BL/6J wild type (WT), BL/6.129P2-IL-10tm1Cgn/J (IL-10-/-) and B6.129S7-Rag1tm1Mom/J (Rag1-/-) mice were purchased from The Jackson Laboratory (Bar Harbor, MA) and bred in a specific pathogen free breeding colony. Mice at 8 – 12 weeks of age were orally inoculated with Tryptone Soy Broth (TSB – vehicle control) or 109 CFU of C. jejuni 11168 in 0.2ml TSB, as described previously21. All animal protocols were approved by the Michigan State University Institutional Animal Care & Use Committee. Colonization by C. jejuni was confirmed and Hematoxylin and eosin stained sections of the ileocecocolic junction were scored as described previously21.

Antibodies for in-vivo neutralization

Anti-IFN-γ (XMG1.2), α-IL-10R (1B1.3A) and α-Thy-1 (30H12) was purchased from Bio-X-Cell, and α-IL-17 (ebioMM17F3) was purchased from eBiosciences. XMG1.2 and ebioMM17F3 injected with 500μg and 200μg i.p. respectively per mouse twice weekly, starting on the day of inoculation. α-IL-10R (1B1.3A) was injected with 1mg/mouse i.p. at the time of inoculation, and weekly thereafter. α-Thy-1.2 (30H12) was injected with 250μg/mouse i.p. twice a week, starting 3 days before inoculation. Rat or mouse IgG (Jackson ImmunoResearch) were used as controls where appropriate.

Immunohistochemical analysis of mononuclear cells

5 μm serial sections were cut, deparaffinized in two changes of xylene and rehydrated in graded alcohol series. Slides were incubated in 1% H202 in TBS and boiled for 15 minutes in citrate buffer (Vector laboratories) for CD3 or incubated at 37°C in Proteinase K (Genemed biotechnologies) for F4/80. Thereafter, sections were blocked for 1 – 3 hours with 1% BSA + 1.5% Goat serum in TBS and then incubated with respective primary antibody (1:500 for CD3ε, 1:50 for F4/80) at 4°C overnight. Slides were then stained with the Vectastain ABC kit (Vector laboratories) for CD3 or rat on mouse HRP polymer kit (Biocare medical) for F4/80 according to the manufacturer's instructions. Non-specific IgG (Vector laboratories) for CD3 or irrelevant isotype control (RTK4530, Biolegend) for F4/80 were used as negative controls. From each section, ten non–overlapping 400× magnification fields were chosen randomly, photographed and positively staining cells were counted using the ImageJ cell counter (N.I.H., Bethesda, MD).

Preparation of colonic lamina propria leukocytes

Lamina propria leukocytes were isolated as previously described 51,52. Briefly, for removal of epithelial cells, the colon was washed; cut into small pieces, and then the pieces were incubated with calcium- and magnesium-free HBSS supplemented with 5% FBS and 5 mM EDTA (Sigma-Aldrich) at 100 rpm at 37°C for 30 min. The tissues were then incubated with RPMI 1640 containing 10% FBS and 0.5 mg/ml collagenase type IV and 0.5mg/ml DNaseI (Sigma-Aldrich) for 1 hour at 37°C with shaking at 150 rpm. The liberated cells were collected by passage through a 70 μm nylon mesh. The isolated cells were separated on a 40/80% discontinuous Percoll gradient (GE Bioscience).

Flow cytometry

The following monoclonal antibodies (eBiosciences or Biolegend) were used: anti-CD3 (clone 145–2C11), anti-CD4 (clone RM 4-5), CD8 (clone 53-67); anti-TCR γδ(clone GL3), CD19 (clone 1D3); anti NKp46 (clone 29A1.4); anti-CD11b (clone M1/70), anti-Gr1 (clone-RB6-8C5), anti-CD90.2 (clone53-2.1) and anti-CD16/CD32 (clone 2.4G2). The cells were preincubated for 20 minutes with anti-CD16/CD32 to block Fc receptors then washed and labeled with appropriate mixture of antibodies or isotype matched controls for 30 minutes, centrifuged at 650g, and resuspended in FACS buffer. To exclude dead/dying leukocytes were gated according to forward and side scatter. For intracellular cytokine staining, cells were restimulated for 4 hours with cell stimulation cocktail (eBioscience) and fixed and permeabilized using fixation and permeabilization solution (eBioscience). All cells were analyzed on a LSRII flow cytometer (BD Biosciences) using FlowJo software (Tree Star).

Enzyme-linked Immunosorbent Assay

All cytokines were measured according to the manufacturer's protocol (Ready-Set-Go ELISA kits, eBioscience). To prepare protein extracts from colon, flash frozen tissue was homogenized with 0.5 ml HBSS 1% Triton X-100 with the mini protease inhibitor cocktail (Roche). The homogenates were centrifuged at 12,000g for 30 min at 4°C, and the supernatants were collected for ELISA. For ex vivo culture, RBC-depleted MLN cells at 5×106 cells/ml or 0.5cm of proximal colon in 0.5ml of RPMI 1640 supplemented with 10% FBS and 100μg/ml penicillin G, 10μg/ml of streptomycin and gentamicin and 5μg/ml amphotericin B were incubated at 37°C for 48 hours. Supernatants were clarified and stored at -70°C. C. jejuni specific antibody ELISA has been described previously21. The following plasma dilutions were used: 1:10 for IgA, 1:50 for IgG1, IgG2c and IgG3, 1:100 for IgM and 1:400 for IgG2b. Only absorbance values more than 2 SD away from mean of negative control were considered positive. GM1 (Sigma) and GD1a (USBio) were used at 2 and 20μg/ml respectively and handled similarly.

Quantitative and reverse transcriptase PCR

RNA was extracted from flash frozen proximal colon samples using RNaeasy plus extraction kit (Qiagen). cDNA was subsequently synthesized by GoScript Reverse Transcriptase kit (Promega). Real-time PCR was performed for the target and hprt genes using Quantifast primers and probe assay (Qiagen) in ABI 7500 PCR machine (Applied Biosystems) or iQ5 iCycler (Bio-Rad). For C. jejuni DNA estimation in feces, DNA was extracted as described previously21. 50ng of fecal DNA was used in Q-PCR reaction with C. jejuni specific gyrA primers53 and iQ SYBR green supermix. C. jejuni DNA in fecal DNA was estimated by running a parallel standard curve of pure C. jejuni DNA.

Splenocyte challenge by gentamicin killing assay

RBC depleted splenocytes from naïve C57BL/6 wt mouse (106 cells/ml) were plated in antibiotic free R10 medium and challenged with indicated C. jejuni strains at multiplicity of infection (M.O.I) of 0.1, 1 or 10. One hour after infection, gentamicin (250μg/ml) was added to all the wells to kill extracellular bacteria. Supernatants were collected after 72 hours for cytokine measurement by ELISA. To obtain adherent cells, splenocytes were plated at 107 cells/ml for 90 minutes upon which the non-adherent cells were washed off.

Epithelial challenge by gentamicin killing assay

1.5×106 Young Adult Mouse Colon (YAMC) cells were grown in permissive media (RPMI 1640 with 5% FBS, ITS and 5 IU/ml IFN-γ) in 24 wells plates at 33°C and 5% CO2. At 80% confluence media is changed to ITS and IFN-γ free RPMI 1640 with 5% FBS and incubated for 18 hours at 37°C. C. jejuni is then added at M.O.I of 100 followed by two hour incubation and three washed in PBS. For measuring invasion cells are further incubated for one hour with 250 μg/ml gentamicin, washed in PBS, lysed in 0.1% Triton X-100 and released bacteria is enumerated by serial dilution. For cytokine measurement cells are incubated for further 24 hours in media containing gentamicin and supernatant is clarified. Sensitivity of all strains to this concentration of gentamicin was also confirmed.

Statistical analysis

All statistical tests were performed in Prism 6.0 (GraphPad Software) and described in figure legends. *p < 0.05, **p < 0.01, ***p < 0.001, ns not significant.

Supplementary Material

Figure S1. A) Intracellular cytokine staining for colon T cells and innate lymphocytes. Representative shown is an infected mouse from day 11 post inoculation. Note that Innate Lymphoid cells were gated as Thy1+CD3-CD19- for ICCS that were confirmed to be negative other lineage markers in the previous cell counting experiments. Staining for IL-17 and IL-22 was performed separately on same cell preparation. B) C57BL/6 IL-10-/- were injected with α-Thy1.2 (clone 30H12) or Rat CIgG. Depletion of Thy-1+ cells was confirmed by α-Thy1.2 (clone53-2.1) staining in blood and colon on day 28 post first injection.

Figure S2. A) C57BL/6 IL-10-/- mice were orally inoculated with given C. jejuni strains and sacrificed at Day 35 post inoculation, or earlier if requiring euthanasia. Mice were bled at time of necropsy, and plasma was analyzed for indicated antibody isotypes for binding to GM1 and GD1a by ELISA.

Suppl Table 1. Spearman correlation values for histologic lesion score and levels of various selected cytokines in the colons of C. jejuni 11168 infected C57BL/6 IL-10-/- mice. n=8-10 mice. Data representative of one of three independent experiments.

Acknowledgments

Projects were funded in whole with federal funds from National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. N01-AI-30058 and Enteric Research Investigational Network Cooperative Research Center (ERIN CRC) grant U19AI090872 to Michigan State University. We thank the Michigan State University Graduate School, Provost's Office, and Vice President for Research and Graduate Studies offices for their matching funds to the ERIN CRC that supported the salary and stipend for Ankit Malik and other contributing doctoral students. We would also like to thank Julia A. Bell for statistical advice and Kathleen A. Hoag for critical review of this manuscript.

Footnotes

Conflict of Interest Statement: No conflict

References

  • 1.CDC. Incidence of laboratory-confirmed bacterial and parasitic infections, and postdiarrheal hemolytic uremic syndrome (HUS), by year and pathogen. 2012;1996 - 2011 http://www.cdc.gov/foodnet/data/trends/tables/table2a-b.html#table-2b. [Google Scholar]
  • 2.Dasti JI, Tareen AM, Lugert R, Zautner AE, Groß U. Campylobacter jejuni: A brief overview on pathogenicity-associated factors and disease-mediating mechanisms. International Journal of Medical Microbiology. 2010;300:205–211. doi: 10.1016/j.ijmm.2009.07.002. [DOI] [PubMed] [Google Scholar]
  • 3.Butzler JP. Campylobacter, from obscurity to celebrity. Clinical Microbiology and Infection. 2004;10:868–876. doi: 10.1111/j.1469-0691.2004.00983.x. [DOI] [PubMed] [Google Scholar]
  • 4.van Spreeuwel JP, et al. Campylobacter colitis: histological immunohistochemical and ultrastructural findings. Gut. 1985;26:945–951. doi: 10.1136/gut.26.9.945. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Blaser MJ. Epidemiologic and clinical features of Campylobacter jejuni infections. Journal of Infectious Diseases. 1997;176:S103–S105. doi: 10.1086/513780. [DOI] [PubMed] [Google Scholar]
  • 6.Tee W, Mijch A. Campylobacter jejuni bacteremia in human immunodeficiency virus (HIV)-infected and non-HIV-infected patients: comparison of clinical features and review. Clinical infectious diseases. 1998;26:91–96. doi: 10.1086/516263. [DOI] [PubMed] [Google Scholar]
  • 7.Gradel KO, et al. Increased Short- and Long-Term Risk of Inflammatory Bowel Disease After Salmonella or Campylobacter Gastroenteritis. Gastroenterology. 2009;137:495–501. doi: 10.1053/j.gastro.2009.04.001. doi: http://dx.doi.org/10.1053/j.gastro.2009.04.001. [DOI] [PubMed] [Google Scholar]
  • 8.Qin HY, et al. Systematic review of animal models of post-infectious/post-inflammatory irritable bowel syndrome. J Gastroenterol. 2011;46:164–174. doi: 10.1007/s00535-010-0321-6. [DOI] [PubMed] [Google Scholar]
  • 9.Garg AX, Pope JE, Thiessen-Philbrook H, Clark WF, Ouimet J. Arthritis risk after acute bacterial gastroenteritis. Rheumatology. 2008;47:200–204. doi: 10.1093/rheumatology/kem339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Yuki N, et al. Association of IgG anti-GD1a antibody with severe Guillain–Barré syndrome. Muscle & Nerve. 1993;16:642–647. doi: 10.1002/mus.880160610. [DOI] [PubMed] [Google Scholar]
  • 11.Vucic S, Kiernan MC, Cornblath DR. Guillain-Barré syndrome: An update. Journal of Clinical Neuroscience. 2009;16:733–741. doi: 10.1016/j.jocn.2008.08.033. [DOI] [PubMed] [Google Scholar]
  • 12.Hughes RA, Cornblath DR. Guillain-Barre syndrome. Lancet. 2005;366:1653–1666. doi: 10.1016/S0140-6736(05)67665-9. [DOI] [PubMed] [Google Scholar]
  • 13.CDC. Gullian Barre Syndrome. 2012 http://www.cdc.gov/flu/protect/vaccine/guillainbarre.htm.
  • 14.Jacobs BC, et al. Campylobacter jejuni infections and anti-GM1 antibodies in guillain-barré syndrome. Annals of Neurology. 1996;40:181–187. doi: 10.1002/ana.410400209. [DOI] [PubMed] [Google Scholar]
  • 15.Islam Z, et al. Guillain-Barré Syndrome-Related Campylobacter jejuni in Bangladesh: Ganglioside Mimicry and Cross-Reactive Antibodies. PLoS ONE. 2012;7:e43976. doi: 10.1371/journal.pone.0043976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Goodfellow JA, et al. Overexpression of GD1a ganglioside sensitizes motor nerve terminals to anti-GD1a antibody-mediated injury in a model of acute motor axonal neuropathy. The Journal of neuroscience. 2005;25:1620–1628. doi: 10.1523/JNEUROSCI.4279-04.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Kieseier BC, et al. Advances in understanding and treatment of immune-mediated disorders of the peripheral nervous system. Muscle & Nerve. 2004;30:131–156. doi: 10.1002/mus.20076. [DOI] [PubMed] [Google Scholar]
  • 18.Ouyang W, Rutz S, Crellin NK, Valdez PA, Hymowitz SG. Regulation and Functions of the IL-10 Family of Cytokines in Inflammation and Disease. Annual Review of Immunology. 2011;29:71–109. doi: 10.1146/annurev-immunol-031210-101312. [DOI] [PubMed] [Google Scholar]
  • 19.Saraiva M, O'Garra A. The regulation of IL-10 production by immune cells. Nat Rev Immunol. 2010;10:170–181. doi: 10.1038/nri2711. [DOI] [PubMed] [Google Scholar]
  • 20.Franke A, et al. Sequence variants in IL10, ARPC2 and multiple other loci contribute to ulcerative colitis susceptibility. Nat Genet. 2008;40:1319–1323. doi: 10.1038/ng.221. doi: http://www.nature.com/ng/journal/v40/n11/suppinfo/ng.221_S1.html. [DOI] [PubMed] [Google Scholar]
  • 21.Mansfield LS, et al. C57BL/6 and Congenic Interleukin-10-Deficient Mice Can Serve as Models of Campylobacter jejuni Colonization and Enteritis. Infection and Immunity. 2006;75:1099–1115. doi: 10.1128/iai.00833-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Mansfield L, et al. Genetic background of IL-10−/− mice alters host–pathogen interactions with Campylobacter jejuni and influences disease phenotype. Microbial Pathogenesis. 2008;45:241–257. doi: 10.1016/j.micpath.2008.05.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Mansfield L, et al. Campylobacter. 3rd. ASM Press; Washington, DC: 2008. Animal models of Campylobacter jejuni infections; pp. 367–379. [Google Scholar]
  • 24.Bell JA, et al. Outcome of infection of C57BL/6 IL-10(-/-) mice with Campylobacter jejuni strains is correlated with genome content of open reading frames up- and down-regulated in vivo. Microbial Pathogenesis. 2013;54:1–19. doi: 10.1016/j.micpath.2012.08.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Bell JA, et al. Multiple factors interact to produce responses resembling spectrum of human disease in Campylobacter jejuni infected C57BL/6 IL-10-/- mice. BMC Microbiology. 2009;9:57. doi: 10.1186/1471-2180-9-57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Bereswill S, et al. Gnotobiotic IL-10−/−; NF-κBEGFP Mice Develop Rapid and Severe Colitis Following Campylobacter jejuni Infection. PLoS ONE. 2009;4:e7413. doi: 10.1371/journal.pone.0007413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Sun X, Threadgill D, Jobin C. Campylobacter jejuni Induces Colitis Through Activation of Mammalian Target of Rapamycin Signaling. Gastroenterology. 2012 doi: 10.1053/j.gastro.2011.09.042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Sun X, Liu B, Sartor RB, Jobin C. Phosphatidylinositol 3-Kinase-γ Signaling Promotes Campylobacter jejuni–Induced Colitis through Neutrophil Recruitment in Mice. The Journal of Immunology. 2013;190:357–365. doi: 10.4049/jimmunol.1201825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Koga M, et al. Anti-GM1 antibody IgG subclass A clinical recovery predictor in Guillain–Barré syndrome. Neurology. 2003;60:1514–1518. doi: 10.1212/01.wnl.0000061615.77865.83. [DOI] [PubMed] [Google Scholar]
  • 30.Reinhardt RL, Liang HE, Locksley RM. Cytokine-secreting follicular T cells shape the antibody repertoire. Nat Immunol. 2009;10:385–393. doi: 10.1038/ni.1715. doi: http://www.nature.com/ni/journal/v10/n4/suppinfo/ni.1715_S1.html. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Stavnezer J. Immunoglobulin class switching. Current Opinion in Immunology. 1996;8:199–205. doi: 10.1016/s0952-7915(96)80058-6. [DOI] [PubMed] [Google Scholar]
  • 32.Deenick EK, Hasbold J, Hodgkin PD. Decision criteria for resolving isotype switching conflicts by B cells. European Journal of Immunology. 2005;35:2949–2955. doi: 10.1002/eji.200425719. [DOI] [PubMed] [Google Scholar]
  • 33.Bai Y, et al. CCL2 recruitment of IL-6-producing CD11b+ monocytes to the draining lymph nodes during the initiation of Th17-dependent B cell-mediated autoimmunity. European Journal of Immunology. 2008;38:1877–1888. doi: 10.1002/eji.200737973. [DOI] [PubMed] [Google Scholar]
  • 34.Bernink JH, et al. Human type 1 innate lymphoid cells accumulate in inflamed mucosal tissues. Nature Immunology. 2013 doi: 10.1038/ni.2534. [DOI] [PubMed] [Google Scholar]
  • 35.Sonnenberg GF, Artis D. Innate lymphoid cell interactions with microbiota: implications for intestinal health and disease. Immunity. 2012;37:601–610. doi: 10.1016/j.immuni.2012.10.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Diefenbach A, Vonarbourg C. Innate lymphocytes induce inflammatory bowel disease. Immunology and Cell Biology. 2010;88:694–696. doi: 10.1038/icb.2010.82. [DOI] [PubMed] [Google Scholar]
  • 37.Cua DJ, Tato CM. Innate IL-17-producing cells: the sentinels of the immune system. Nature Reviews Immunology. 2010;10:479–489. doi: 10.1038/nri2800. [DOI] [PubMed] [Google Scholar]
  • 38.Spits H, Di Santo JP. The expanding family of innate lymphoid cells: regulators and effectors of immunity and tissue remodeling. Nature Immunology. 2010;12:21–27. doi: 10.1038/ni.1962. [DOI] [PubMed] [Google Scholar]
  • 39.Cherrier M, Ohnmacht C, Cording S, Eberl G. Development and function of intestinal innate lymphoid cells. Current Opinion in Immunology. 2010 doi: 10.1016/j.coi.2012.03.011. [DOI] [PubMed] [Google Scholar]
  • 40.Sonnenberg GF, Monticelli LA, Elloso MM, Fouser LA, Artis D. CD4+ Lymphoid Tissue-Inducer Cells Promote Innate Immunity in the Gut. Immunity. 2011;34:122–134. doi: 10.1016/j.immuni.2010.12.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Buonocore S, et al. Innate lymphoid cells drive interleukin-23-dependent innate intestinal pathology. Nature. 2010;464:1371–1375. doi: 10.1038/nature08949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Caramaschi P, et al. Anti-TNFalpha blockers, autoantibodies and autoimmune diseases. Joint Bone Spine. 2009;76:333–342. doi: 10.1016/j.jbspin.2008.12.008. doi: http://dx.doi.org/10.1016/j.jbspin.2008.12.008. [DOI] [PubMed] [Google Scholar]
  • 43.Lozeron P, D C, L C, A D. Long-term course of demyelinating neuropathies occurring during tumor necrosis factor-α–blocker therapy. Arch Neurol. 2009;66:490–497. doi: 10.1001/archneurol.2009.11. [DOI] [PubMed] [Google Scholar]
  • 44.Tristano AG. Neurological adverse events associated with anti-tumor necrosis factor alpha treatment. Journal of neurology. 2010;257:1421–1431. doi: 10.1007/s00415-010-5591-7. [DOI] [PubMed] [Google Scholar]
  • 45.Zhang M, et al. Association study between an outbreak of Guillain-Barre syndrome in Jilin, China, and preceding Campylobacter jejuni infection. Foodborne Pathogens and Disease. 2010;7:913–919. doi: 10.1089/fpd.2009.0493. [DOI] [PubMed] [Google Scholar]
  • 46.Kalra V, et al. Association of Campylobacter jejuni Infection with Childhood Guillain-Barré Syndrome: A Case-Control Study. Journal of Child Neurology. 2009;24:664–668. doi: 10.1177/0883073808325649. [DOI] [PubMed] [Google Scholar]
  • 47.Bax M, et al. Campylobacter jejuni lipooligosaccharides modulate dendritic cell-mediated T cell polarization in a sialic acid linkage-dependent manner. Infection and Immunity. 2011;79:2681–2689. doi: 10.1128/IAI.00009-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Huizinga R, et al. Sialylation of Campylobacter jejuni Lipo-Oligosaccharides: Impact on Phagocytosis and Cytokine Production in Mice. PLoS ONE. 2012;7:e34416. doi: 10.1371/journal.pone.0034416. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Klaas M, et al. Sialoadhesin Promotes Rapid Proinflammatory and Type I IFN Responses to a Sialylated Pathogen, Campylobacter jejuni. The Journal of Immunology. 2012;189:2414–2422. doi: 10.4049/jimmunol.1200776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Weigmann B, et al. Isolation and subsequent analysis of murine lamina propria mononuclear cells from colonic tissue. Nature Protocols. 2007;2:2307–2311. doi: 10.1038/nprot.2007.315. [DOI] [PubMed] [Google Scholar]
  • 51.Sanos SL, et al. RORγt and commensal microflora are required for the differentiation of mucosal interleukin 22–producing NKp46+ cells. Nature Immunology. 2008;10:83–91. doi: 10.1038/ni.1684. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Wilson DL, Abner SR, Newman TC, Mansfield LS, Linz JE. Identification of Ciprofloxacin-ResistantCampylobacter jejuni by Use of a Fluorogenic PCR Assay. Journal of clinical microbiology. 2000;38:3971–3978. doi: 10.1128/jcm.38.11.3971-3978.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1. A) Intracellular cytokine staining for colon T cells and innate lymphocytes. Representative shown is an infected mouse from day 11 post inoculation. Note that Innate Lymphoid cells were gated as Thy1+CD3-CD19- for ICCS that were confirmed to be negative other lineage markers in the previous cell counting experiments. Staining for IL-17 and IL-22 was performed separately on same cell preparation. B) C57BL/6 IL-10-/- were injected with α-Thy1.2 (clone 30H12) or Rat CIgG. Depletion of Thy-1+ cells was confirmed by α-Thy1.2 (clone53-2.1) staining in blood and colon on day 28 post first injection.

Figure S2. A) C57BL/6 IL-10-/- mice were orally inoculated with given C. jejuni strains and sacrificed at Day 35 post inoculation, or earlier if requiring euthanasia. Mice were bled at time of necropsy, and plasma was analyzed for indicated antibody isotypes for binding to GM1 and GD1a by ELISA.

Suppl Table 1. Spearman correlation values for histologic lesion score and levels of various selected cytokines in the colons of C. jejuni 11168 infected C57BL/6 IL-10-/- mice. n=8-10 mice. Data representative of one of three independent experiments.

NIHMS531784-supplement-supplement_1.pdf (2.4MB, pdf)

RESOURCES