CD4+ T-cell reactivation and interleukin (IL)–2 release are responsible for acute gastrointestinal symptoms when patients with celiac disease are exposed to gluten, according to investigators.
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Although T cells have been well studied in previous celiac disease research, clinical symptoms after acute gluten exposure have never been linked with specific cytokine changes, reported lead author Gautam Goel, PhD, of Massachusetts General Hospital in Boston, and colleagues.
“If treated [celiac disease] patients, i.e., those following a strict [gluten-free diet], are exposed to gluten-containing food, they typically suffer from gastrointestinal reactions occurring 1 to 2 hours after the gluten exposure,” the investigators wrote in Science Advances. “There is currently no explanation for the acute gluten-induced symptoms seen in treated [celiac disease] patients.”
The current study was prompted by two phase 1 trials involving the therapeutic vaccine Nexvax2, which uses peptide fragments of gluten proteins to desensitize celiac patients to gluten, the investigators explained. During those trials, intradermal injections of Nexvax2 above a certain dose threshold led to gastrointestinal symptoms within 2-5 hours, but not injection site reactions, which would have been indicative of a cutaneous response to recall antigen.
“Our observations from these phase 1 studies led us to hypothesize that cytokine release occurs following natural gluten exposure and could be used to implicate which arms of the immune system drive early symptoms.”
Of the 28 patients in the two trials, all underwent intradermal testing, while 19 also participated in an oral gluten challenge. Following intradermal injection of gluten peptides, patients exhibited gastrointestinal symptoms, along with coordinated elevations of least 15 plasma cytokines; most significantly IL-2, MCP-1, IL-8, IL-10, MIP-1beta, IP-10, and eotaxin. The first cytokines to respond to injection were IL-2 and IL-8, rising within 2 hours, prior to symptoms. At 4 hours, when symptoms were present, peak IL-2 elevations were most dramatic, with a 272-fold elevation, followed by IL-8 (11-fold) and IL-10 (1.2-fold).
“IL-2 is both the earliest and most sensitive marker for the coordinated cytokine release that was almost universal in HLA-DQ2.5 + [celiac disease] patients administered gluten peptides,” the investigators wrote.
Similar to intradermal testing, oral challenge with gluten caused IL-2, IL-8, and IL-10 to elevate within 2 hours, and peak within 4-6 hours. Again, IL-2 was most sensitive, with a 15-fold increase at 4 hours. This increase in IL-2 correlated with IL-8 and IL-10 elevations, although IL-2 increases were at least six times greater than the other two cytokines.
“Together, the serum cytokine profile following gluten ingestion is less prominent but qualitatively similar and over a corresponding time course to that after injecting gluten peptides, which is consistent with activated CD4+ T cells being the driver of cytokine release in both scenarios,” the investigators wrote.
Further testing showed that, after gluten challenge, plasma levels of IL-2, IL-8, and IL-10 negatively correlated with duodenal villous height-to-crypt depth ratios. In addition, high levels of IL-2 correlated with severe nausea and vomiting, adding to the evidence that celiac symptoms were linked with specific cytokine elevations.
“The link between immune activation and symptoms was further strengthened by showing that postdose symptoms and cytokine release were both lessened after three weekly doses and absent after 16 twice-weekly injections of gluten peptides,” the investigators wrote. “These findings are consistent with the difference in severity of symptoms after gluten ingestion compared to gluten peptide injection being related to potency of the antigen challenge and T-cell activation measured by circulating IL-2 concentration at 4 hours.”
Even though IL-2 elevations appeared to drive celiac symptoms, the source of IL-2 was initially unknown. “Activated T cells are the primary source of IL-2, but [dendritic cells] can also secrete IL-2 following ligation of specific pathogen recognition receptors; mast cells also secrete IL-2 following exposure to IL-33 or IL-9,” the investigators explained. Still, CD4+ T cells are known to be key players in celiac disease, and the timing and magnitude of IL-2 release made T cells the most likely candidates. To test this hypothesis, the investigators collected blood from patients 6 days after gluten food challenge and incubated these samples for 24 hours with gluten peptides. Results of this test suggested that gluten-specific CD4+ T cells were the most likely source of IL-2.
The connection between particular cytokines and gastrointestinal symptoms is now supported with evidence; however, the investigators pointed out that a relationship between cytokines and other symptoms of celiac disease remains to be seen. “Whether cytokines elevated in blood after injecting gluten peptides or ingesting gluten have any direct extraintestinal effects is unclear,” the investigators wrote. “Fatigue, headache, and ‘brain fog’ are the commonly reported extraintestinal symptoms in [celiac disease] patients. However, symptoms being focused on the upper gastrointestinal tract suggest that cytokines increased in blood have clinical and immunological effects that selectively affect the tissue from which they originate.”
“Future studies should test whether cytokine concentrations are substantially higher in gut mucosal tissue than in blood after gluten challenge or injection of gluten peptides and determine whether alterations in local cytokine levels are matched by immune and inflammatory cell infiltration,” the investigators wrote.
The study was supported by the University of Chicago Celiac Disease Center and the University of Oslo KG Jebsen Coeliac Disease Research Centre. The investigators reported additional relationships The investigators reported additional relationships with several government and nonprofit organizations. Multiple investigators are employees of ImmusanT, which is developing Nexvax2.
SOURCE: Goel G et al. Science Advances. 2019 Aug 7. doi: 10.1126/sciadv.aaw7756.