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Intact Regulatory T-Cell Function but Defective Generation of IL–17A-Producing CD4+ T Cells in XIAP Deficiency

Gurram, Bhaskar*; Hammelev, Erin; Syverson, Grant; Haribhai, Dipica; Yan, Key§; Simpson, Pippa§; Salzman, Nita*; Verbsky, James W.

Journal of Pediatric Gastroenterology and Nutrition: August 2016 - Volume 63 - Issue 2 - p 218–225
doi: 10.1097/MPG.0000000000001122
Original Articles: Gastroenterology

Objective: X-linked inhibitor of apoptosis (xIAP) deficiency is a primary immune deficiency disorder associated with hemophagocytic lymphohistiocytosis. About 17% of xIAP-deficient patients present with very early onset severe colitis with high mortality. We hypothesized that xIAP deficiency leads to defective generation and/or survival of T regulatory cells (Treg) through its involvement in transforming growth factor-β signaling.

Methods and Results: We used a T-cell transfer model of chronic colitis and observed a mild increase in colitis severity induced by naïve CD4+ T cells from xIAP0/ mice compared with colitis induced by naïve CD4+ T cells from WT mice. We did not observe any significant difference in the induction of Treg cells in these studies. We next tested whether xIAP is required for Treg cell function by co-transferring xIAP−/0 or WT Treg cells with naïve WT CD4 cells in this model. We demonstrate that XIAP-deficient Treg cells were able to prevent disease similarly to WT Treg cells. In these experiments we, however, found a significantly decreased percentage of IL–17A-producing CD4+ T cells in mice receiving Tregs from xIAP0/ mice.

Conclusions: xIAP appears dispensable for the generation of induced Treg cells as well as function of natural Treg cells. There appeared to be a role of xIAP in generation of IL–17-producing cells from either naïve CD4+ T cells or Treg cells. Further research is needed to explore the role of xIAP in generation of IL–17-producing cells.

*Department of Pediatrics, Division of Gastroenterology

Department of Pediatrics, Division of Rheumatology, Medical College of Wisconsin, Milwaukee

Pediatric Rheumatology Division, University of Wisconsin School of Medicine and Public health, Madison

§Department of Pediatrics, Section of Quantitative Health Sciences, Medical College of Wisconsin, Milwaukee.

Address correspondence and reprint requests to Bhaskar Gurram, MBBS, MD, Medical College of Wisconsin, Milwaukee, WI 53226 (e-mail:

Received 2 March, 2015

Accepted 16 January, 2016

J.W.V. was supported by National Institutes of Health (NIH) KO8AI072023-01.

The authors report no conflicts of interests.

What Is Known

  • About 1/5th of patients with X-linked inhibitor of apoptosis protein deficiency present with colitis.
  • In most patients, colitis is early onset and refractory to conventional therapies.
  • Some reports showed low regulatory T-cell numbers in patients with X-linked inhibitor of apoptosis protein deficiency.
  • X-linked inhibitor of apoptosis protein is involved in antiapoptotic function, nucleotide-binding oligomerization domain signaling, and transforming growth factor-β signaling; however, which of these pathways are involved in the pathogenesis of colitis is unknown.

What Is New

  • In a mouse model of experimental colitis, X-linked inhibitor of apoptosis protein appears to be dispensable for generation of regulatory T cells.
  • There appears to be a role of X-linked inhibitor of apoptosis protein in the generation of IL–17-producing cells.

X-linked inhibitor of apoptosis protein (xIAP) is an ubiquitously expressed inhibitor of apoptosis (1). Studies have shown a role for xIAP in nucleotide-binding oligomerization domain containing-1 (NOD1) and NOD2 signaling (2–4), transforming growth factor (TGF)-β signaling (5,6), and ubiquitin ligase activity (7). xIAP deficiency leads to X-linked lymphoproliferative disease type 2, typically associated with hemophagocytic lymphohistiocytosis in response to viral infections (8–11). We described a patient with early onset fistulizing inflammatory bowel disease due to a point mutation in xIAP (12). About 17% of the patients with xIAP deficiency present with severe early onset colitis that is uniformly refractory to conventional inflammatory bowel disease (IBD) treatment and associated with high mortality (8,10,11,13).

How xIAP prevents inflammatory responses in the gastrointestinal (GI) tract is unknown, but several mechanisms can be proposed based on the known functions of xIAP. xIAP is an ubiquitously expressed inhibitor of apoptosis that binds caspases and inhibits their activation (1). Thus a deficiency of xIAP could result in enhanced apoptosis of Treg cells (regulatory T cells) or other regulatory populations leading to the development of IBD. Studies also demonstrate a role for xIAP in NOD1 and NOD2 signaling (2), TGF-β signaling (5,6), and ubiquitin ligase activity (7). xIAP directly interacts with both NOD2 and receptor-interacting serine threonine kinase 2 and is required for the activation of NF-κB and cytokine production in response to NOD1 and NOD2 ligands (23,4). Mutations in NOD2 are implicated in IBD development, although the mechanism remains unclear. NOD2 ligation by muramyl dipeptide inhibits Toll-like receptor responses (14,15). NOD2 is highly expressed in paneth cells and regulates defensin expression (16). NOD2-deficient mice exhibit greater bacterial loads in the colon, enhanced GI permeability, and increased bacterial translocation (17,18). In addition, challenge of these mice with enteric pathogens resulted in greater systemic bacterial burdens (19). Finally, NOD2 mediates the production of IL-10 from dendritic cells that promote the survival/expansion of Treg cells (20). Interestingly, NOD1/NOD2 double-deficient mice exhibit decreased IL-17 and IL-22 production following challenge with colitogenic bacteria, whereas single knockouts demonstrated no affect (21). Thus, defective NOD1/NOD2 signaling due to xIAP deficiency could have significant impact on inflammatory responses in the GI tract. Importantly, because NOD1 and NOD2 signaling are dependent on xIAP, this may explain why colitis is more severe in xIAP deficiency compared with NOD2 deficiency.

xIAP also binds the TGF-β type I receptor, mediates the ubiquitination of TGF–β-activated kinase 1 (TAK1), and facilitates the formation of complexes between TAK1-binding protein 1 and IκB kinase enabling TGF-β to activate p65/RelA (22,23). TGF-β has numerous roles in immune regulation, including the generation and function of Treg cells (24–26). TGF-β is also implicated in the generation of TH17 cells, and there appears to be a reciprocal relation between Treg cells and TH17 generation in response to TGF-β, with the later favored if IL-6 is present (27). IL-22 from TH17 cells is known to enhance the epithelial barrier function, and a trial of anti-IL17 antibody in patients with moderate to severe Crohn disease was ineffective and associated with adverse outcomes (28). Thus, TGF-β has numerous possible roles in mucosal immune regulation through the induction/function of Treg cells and the production of TH17 cells that promote maintenance of epithelial barrier function through IL-22 production (21,29,30).

Herein, we used the mice with xIAP deficiency in a T cell transfer model of colitis to investigate the role of xIAP in regulatory T-cell survival and function. The xIAP−/0 mouse phenotype was originally described to have no defects in apoptotic pathways, possibly from compensatory mechanisms of other inhibitors of apoptosis (IAPs) (31). xIAP−/0 mice have been shown to have greater disease burden from intracellular bacterial pathogens (32), as well as increased apoptosis to murine herpes virus infections (33). These studies suggest that when challenged xIAP−/0 mice have abnormal immune regulation. We hypothesized that xIAP deficiency leads to defective Treg cell and/or TH17 cell survival and/or function.

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xIAP/0 mice were obtained from the Mutant Mouse Regional Resource Center and screened for the absence of xIAP as described previously (5,31). Rag1/ mice were obtained from the Jackson laboratory. IL10GFP reporter mice were obtained from Christopher Karp (34). All of the mice used for experiments were fed autoclaved water cohoused to control for microbiome effect. All of the mice were of C57BL/6 background.

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Colitis Model

IL10GFP mice were crossed with xIAP/0 mice to generate xIAP/0IL10GFP reporter mice. Pooled splenocytes and lymph node cells from xIAP/0 or WT mice were stained for CD4, CD25, and CD45RB. All of the sorting was done on a FACSAria (BD Biosciences, East Rutherford, NJ). Colitis was induced in 6–8-week-old RAG1/ C57BL6 mice by i.p. injection of 4 × 105 naïve T cells (CD4+CD25CD45RBhigh) suspended in 1 mL of PBS. Mice were weighed before injection and then twice weekly following the injections. In some experiments, 2 × 105 purified Treg cells (CD4+CD45RBlowCD25hi) were given by i.p. injection in combination with naïve CD4+ T cells (prevention experiments). Mice were analyzed if they lost 20% weight from base line or if they looked moribund as assessed by the animal care facility personnel. Mice that survived up to 120 days without getting sick were also sacrificed and analyzed.

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Analytical Flow Cytometry

Lymphocytes were isolated from mesenteric lymph node (MLN) and spleen. Cells were stained with antibodies to CD4, TCR, CD25, CD62L, and CD44. CD4 cells were analyzed to determine the percent naïve CD4 cells (CD62Lhi/CD44lo) and effector/memory cells (CD62Llo/CD44hi) (10,35). Flow cytometry was performed on a custom 4-laser LSRII and the data analyzed with BD FACSDiva software (BD Biosciences).

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Cytokine Analysis

After isolating cells from MLN and spleen, cells were activated with PMA (50 ng/mL) (Sigma) and ionomycin (1 nM) (Calbiochem) for 4 to 5 hours. For intracellular staining for cytokines and FoxP3 Golgiplug (BD) at 1 μl/mL was added along with PMA and ionomycin. These cells were then stained at 4°C with fluorescent conjugated antimouse antibodies against CD4 and TCR, washed with FACS buffer, and fixed overnight with 1% paraformaldehyde (PFA) at 4°C. Cells were washed with 1x PBS and FACS buffer and then permeabilized 1 hour in 0.1% Triton-X. Intracellular staining for TNF-α, IL-17, and IFN-γ at recommended concentrations was performed at 4°C for 30 minutes. Cells were washed and then fixed with 1% PFA. IL-10 was detected by GFP staining. A 4-laser custom LSRII was used to collect the data, and BD FACSDiva (BD Biosciences) software was used for analysis.

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Foxp3 Analysis

Lymphocytes were isolated from MLN and SPL. Cells were stained at 4°C for CD4, washed with FACS buffer, and fixed overnight with 1% PFA at 4°C. Cells with washed with 1x PBS, washed again with FACS buffer, and permeabilized 1 hour in 0.1% Triton-X. Intracellular staining for Foxp3 was performed at 4°C for 4 hours. Cells were washed with FACS buffer and fixed with 1% PFA. A 4-laser custom LSRII was used to collect the data, and BDFACS Diva software (BD Biosciences) was used for analysis.

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Complete colons were fixed in formalin, processed, and stained with hematoxylin and eosin. Blinded sections from the entire colon were examined by a pathologist, and large intestine colitis scores were determined for the following inflammatory changes on a 4-point semiquantitative scale with 0 representing no colitis, 1 representing mild colitis (total sub-score 1–5), 2 representing moderate colitis (sub-score ranging from 6–10), and 3 representing severe colitis (sub-score ranging from 11–17) (36,37). Histopathological analysis was performed using the following scoring criteria: degree of inflammation in lamina propria (score 0–3); goblet cell loss (score 0–2); abnormal crypts (score 0–3); presence of crypt abscesses (score 0–1); mucosal erosion and ulceration (score 0–1); submucosal spread to transmural involvement (score 0–3); number of neutrophils counted at 40x magnification (score 0–4). Total histopathological score is calculated by combining the scores for each of the 7 parameters for a maximum score of 17.

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Statistical Analysis

Lymphocyte numbers and lymphocyte percentages were compared by a 2-tailed nonparametric Mann-Whitney test. Random coefficient models with quadratic functions and unstructured variance covariance matrices were used to compare the weight loss over time for the different groups. Kaplan-Meier curves with a Wilcoxon log-rank test were used to compare the survival times between 2 groups. A P < 0.05 was considered significant. Software used was SAS version 9.4 (SAS Institute Inc, Cary, NC).

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Colitis Induced by Naive CD4+ T Cells From xIAP−/0 Mice Leads to Earlier Weight Loss Compared With WT Mice

We transferred 4 × 105 naïve (CD45RBhi) CD4+ T cells into Rag1/ mice to induce colitis. We observed a trend toward increased weight loss in the first 56 days in mice receiving xIAP/0 CD4+ T cells (Fig. 1A). Following these mice longer showed that mice receiving xIAP/0 CD4+ T cells exhibitied a mild but significant decrease in survival compared with those with colitis induced by CD4+ T cells from WT mice (Fig. 1B). More than 90% of mice succumbed to colitis in either groups. There was, however, no difference in the colitis scores observed on histololgical scoring (Fig. 1C).



We then isolated lymphocytes from mesenteric lymph nodes from sacrificed mice and determined the percentage of naïve, central memory, and effector memory cells. The gating strategy used in this study and the absolute number of cells isolated from the mesenteric lymphnodes are depicted in Fig. 2. WT C57BL/6 mice were used as staining controls. We examined intracellular cytokine expression and Treg cell percentages. All of the mice with colitis had significantly increased percentages of effector memory T cells in MLN (Fig. 3A). We did not observe any differences in proportion of naïve, central memory, or effector memory T-cell percentages in MLN between Rag1−/− mice receiving WT or xIAP−/0 CD4 cells. The proportions of CD4+ T cells positive for IFN-γ, TNF-α, and IL-17A were increased compared with control mice in both groups and were comparable between the WT and xIAP/0 groups (Fig. 3B). Finally, we did detect a decrease in percentage of Treg cells (CD25+, FoxP3+) as well as IL-10 positive CD4+ T cells in the MLN (Fig. 3C) in mice with colitis compared with healthy WT controls. There was, however, no significant difference between the 2 experimental groups.





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Regulatory T Cells From xIAP−/0 Mice Are Capable of Preventing Colitis Induced by WT Naïve CD4+ T Cells, but a Defect in IL-17 Generation Was Detected

We next examined the ability of Treg cells from WT or xIAP/0 mice to prevent colitis induced by WT naïve CD4+ T cells. In this experiment, we simultaneously transferred 4 × 105 WT naïve CD4+ T cells and 2 × 105 WT or xIAP/0 Treg cells. Transferring WT Tregs prevents the weight loss and reduces colitis scores, and xIAP/0 Treg were similarly effective (Fig. 4A). At sacrifice, colitis severity scores were similar in both groups (Fig. 4B), indicating that xIAP/0 Treg cells are equally capable of preventing colitis in this model.



We then isolated MLN lymphocytes from sacrificed mice and looked for proportion of CD4+ T cells positive for cytokines and also for Treg cell markers. When Treg cells from WT or xIAP/0 mice were transferred with naïve CD4+ T cells, we detected a decrease in percent of CD4 cells expressing TNF-α compared with mice receiving no Treg cells (Fig. 5A). IFN-γ levels were reduced but not to a significant level. We did not find a statistical significant difference in TNF-α or IFN–γ-positive CD4+ T cells when mice were rescued with WT or xIAP−/0 Treg cells. We did detect an increased percentage of IL-10, CD25, and FoxP3-positive CD4+ cells in mice receiving Treg cells, but there was no difference between mice that received WT or xIAP/0 Treg cells (Fig. 5B). Interestingly, when xIAP/0 Treg cells were cotransferred with naïve WT CD4+ T cells, there was a significantly lower percentage of IL-17+ CD4+ T cells (P = 0.0076).



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These studies examined the role of xIAP in the function of CD4+ cells and Treg cells in a lymphopenic model of inflammatory colitis. In this model, successful treatment of established disease depends upon the generation of Treg cells from the conventional T-cell pool (38). Indeed, the use of CD4+ T cells unable to induce FoxP3 results in greater severity of colitis (38). Whether induction of FoxP3 is dependent on TGF-β in vivo is not known. We hypothesized that defective TGF-β signaling in xIAP/0 mice may result in the inability to induce FoxP3 in this model. We show that transfer of naïve CD4+ cells from xIAP/0 mice resulted in similar percentage of induced FoxP3+ Treg cells, as well as similar percentages of inflammatory cytokine expressing CD4 T cells compared with mice receiving WT CD4 T cells. Because all of the mice were examined once they reached a moribund state, it is possible that kinetic differences in the survival of Treg cells could have been missed, as suggested by the Kaplan-Meier survival curves. We looked at the in vitro induction of Treg cells from Naïve CD4 T cells from xIAP−/0 and WT mice. Based on our preliminary results we did not find differences. It is also possible that C57BL/6 mouse line is not the best model for studying the differences in induced Treg cell numbers and function, because C57BL/6 mice produce fewer induced Treg cells in this model compared with BALB/c mice (Dipica Haribhai and Calvin Williams, unpublished observation).

When the ability of WT or xIAP-deficient Treg cells to prevent colitis was tested, we observed that xIAP-deficient Treg cells were equally proficient in preventing colitis compared with Treg cells from wild type mice. We detected increased percentages of FoxP3+, IL-10+, and CD25+ CD4+ T cells compared with mice not receiving Treg cells, but there was no observable difference between the 2 experimental groups. This argues that Treg cell survival and function are preserved in the setting of xIAP deficiency. This is possible if xIAP function is not needed for survival of Treg cells, or if redundant factors such as cIAP1 and cIAP2 are able to replace the function of xIAP. This is in fact supported by the work done by Harlin et al (31), in which they showed an increase in cIAP1 and cIAP2 in xIAP-deficient mice. Alternatively, these results could argue that Treg generation is not influenced by the XIAP-mediated regulation of TGF-β.

In the prevention model, we observed a significantly reduced frequency of IL-17+ CD4+ T cells in mesenteric lymph nodes of Rag1−/− mice that received naïve WT CD4+ T cells and Treg cells from xIAP/0 mice. This can be interpreted in 2 ways. First, the prevention experiments greatly improved but did not eliminate the colitis as indicated by colitis scores. Similarly, IFN-γ levels were not significantly changed, but did improve. Thus there could be some ongoing inflammation and IL-17 production in these experiments. This would, however, indicate that xIAP/0 Tregs have greater suppressive function compared with WT Tregs in controlling IL-17 expression. Alternatively, these data could suggest that xIAP deficiency in Treg cells leads to abnormal homeostasis of IL-17. Although IL-17 has important roles in autoimmunity and immune pathology, it is also important in normal immune function. Although we are unable to tell if the source of IL–17-producing cells is from the Treg or naïve CD4+ T cells, it is possible that the result of loss of plasticity of Treg cells to convert to IL–17-producing CD4+ T cells. This potentially could be explained by the role of xIAP in TGF-β signaling, because TGF-β has been shown to induce TH17 cells (39). A second possibility is that some function of Treg cells that is dependent on xIAP signaling results in an environment that prevents the expression of IL-17 in conventional T cells. This difference in the IL–17-producing T cells could also arise from differences in the microbiome (40) though this is unlikely in co-housed mice.

X-linked inhibitor of apoptosis is a ubiquitous protein expressed in all cells. It is a highly conserved antiapoptosis protein with some redundant role in regards to prevention of apoptosis along with cIAP1 and cIAP2. But it was shown to have a unique role in immune regulation because defects in xIAP cause early onset colitis (8,10,11,13). Evaluation of a patient with early onset colitis due to xIAP deficiency showed a reproducibly low Treg percentage in peripheral blood. We did not confirm these findings in these studies because there appeared to be no defect in survival of xIAP-deficient Treg cells compared with WT cells in this model of colitis. Alternatively, this patient has a point mutation in xIAP that did not prevent expression (12) whereas the xIAP mice used here exhibit complete lack of xIAP protein. If there was redundancy in the xIAP proteins it is possible that our patient expressed a protein that prevented the other IAP family members for substituting for xIAP.

It is also possible that this model of colitis may not reflect the colitis seen in humans with xIAP deficiency. This model is a chronic colitis model dependent on the generation of pathogenic CD4 cells. This model does not test the role of xIAP in nonlymphoid cells (dendritic cells and other APCs), or a relatively new class of cells known as innate lymphoid cells. It was demonstrated by Gerart et al (41) that human invariant natural killer T and mucosal-associated invariant T cells exhibit a promyelocytic leukemia zinc finger protein-dependent proapoptotic propensity that is counterbalanced by xIAP. This would explain some of the phenotypic manifestations of xIAP deficiency. Mucosal-associated invariant T cells are shown to be important in innate immune defense against bacteria (42) at the level of mucosal surfaces, and this potentially would explain the propensity to develop colitis. Given the role of xIAP in NOD2 signaling, it is possible that the cause of colitis in xIAP deficiency is the inability of innate lymphoid cells or APCs to respond to bacteria in the gut and prevent bacterial translocation. It is interesting that our patient had complete resolution of colitis when ileum was diverted, arguing that the presence of bacteria was critical to the development of colitis (12). This is further supported by studies that demonstrated a critical role for NOD1/NOD2 signaling in the early control of colitigenic bacteria, but it had no effect on the chronic phase of colitis (21). Future studies will be needed to test the role of xIAP in APCs and innate lymphoid cells.

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In a T-cell transfer model of chronic colitis in C57BL/6 mice, XIAP did not seem to influence the function of regulatory T cells. xIAP may, however, play a role in generation of IL–17-producing cells from either naïve CD4+ T cells or Treg cells. Further research is needed to explore the role of xIAP in generation of IL–1-producing cells.

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We take for granted our abilities to visually examine the GI tract, yet like all progress in science, it evolved gradually through the inspirations of many. In 1805, Philipp Bozzini (1773–1809) described an apparatus he called a lichtleiter (light guider) that consisted of a candle, a reflecting mirror, and a cannula for examining orifices, particularly the urethra and rectum. It was largely ignored until 50 years later when Antoine Desormeaux (1815–1894) improved the apparatus by employing a gas arc lamp* that gave a more reliable, strong light.

George Kelling (1866–1945) of Dresden, in 1901, injected air into the abdomen of a dog that resulted in a pneumoperitoneum, and inserted a cystoscope to study the dog's viscera, a procedure that was referred to as “closed cavity” endoscopy. Ten years later, Swede Han Christian Jacobaeus (1879–1937) published his experiments with human laparoscopy, “The Possibilities for Performing Cystoscopy in Examination of Serous Cavities,” that introduced the revolutionary laparoscope as a diagnostic and operative tool.

In 1957, Basil Hirschowitz (1925–2013) from the University of Alabama and physicist Larry Curtiss from the University of Michigan produced the first fiberoptic prototype. By 1970, the first reports of pediatric endoscopy appeared.



The Desormeaux endoscope (1865). Wikimedia Commons.

*In 1805, Cornish chemist Humphry Davy (1778–1829) first described the carbon-arc light.

—Contributed by Angel R. Colón

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experimental colitis; regulatory T cells; X-linked inhibitor of apoptosis protein

© 2016 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology,