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. 2017 Jul 6;102(3):915–923. doi: 10.1189/jlb.3A1216-527R

IL‐23 restoration of Th17 effector function is independent of IL‐6 and TGF‐β in a mouse model of alcohol and burn injury

Xiaoling Li 1,2,3, Abigail R Cannon 1,2,3,4,5, Adam M Hammer 1,2,5, Niya L Morris 1,2,5, Mashkoor A Choudhry 1,2,3,4,5,✉
PMCID: PMC6608048  PMID: 28684599

Short abstract

IL‐6 and TGF‐β do not appear to influence IL‐23‐mediated restoration of Th17 effector cytokines after ethanol and burn injury.

Keywords: T cell, trauma, cytokines, aryl hydrocarbon receptor

Abstract

T cells play a critical role in host defense against intestinal bacteria. We have shown that ethanol combined with burn injury suppresses Peyer's patch (PP) Th17 cytokines 1 d after injury. We assessed the mechanism of suppressed Th17 effector functions. Mice were gavaged with ethanol 4 h before burn injury and euthanized 1, 3, and 7 d after injury. Mesenteric lymph nodes (MLNs), PPs, and spleen Th1 and Th17 cytokines were assessed. A significant decrease in IL‐17, IL‐22, IL‐2, and IFN‐γ were observed in all 3 lymphoid organs 1 and 3 d after injury. We used splenic cells to study the role of IL‐6, IL‐23, TGF‐β, and aryl hydrocarbon receptor (AHR) in suppressing Th17 cytokines. We also assessed whether the AHR agonist 6‐formylindolo (3, 2‐b) carbazole (FICZ) modulates Th17 cytokines. We found a significant decrease in IL‐6 and TGF‐β after ethanol and burn; IL‐23 was undetectable. The reconstitution of IL‐23 in culture medium increased IL‐17 by 2‐fold and IL‐22 by 20‐fold in cells from burn ethanol mice. The restoration of IL‐6 and TGF‐β combined did not influence the release of Th17 cytokines. We observed that AHR was necessary for IL‐23 restoration of IL‐22 after ethanol and burn injury. The AHR agonist FICZ enhanced IL‐22, but not IL‐17. None of these treatments influenced the release of Th1 cytokines. Together, these results suggest that IL‐23 plays a critical role in regulation of Th17 cytokines. Furthermore, IL‐6 and TGF‐β do not appear to influence IL‐23‐mediated restoration of Th17 cytokines after ethanol and burn injury.

Abbreviations

AHR

aryl hydrocarbon receptor

FICZ

6‐formylindolo (3, 2‐b) carbazole

IRF

interferon regulatory factor

MLN

mesenteric lymph node

PP

Peyer's patch

r

recombinant

ROR

retinoic acid‐related orphan receptor

RORC

ROR type C

TBSA

total body surface area

Introduction

Despite advances in the intensive care units, infection remains the leading cause of multiple organ failure after a burn or other major traumatic injury. The treatment of these injuries becomes increasingly difficult when they are accompanied by prior ethanol consumption. Nearly half of all patients with burns or other traumas have been shown to have ethanol in their blood at the time of hospital admission [1, 2, 3, 4, 5–6]. Several studies have shown that intoxication further worsens the pathology associated with burn and trauma [1, 2, 3–4, 7, 8, 9–10]. These patients exhibit higher morbidity and mortality than patients with burns who are not intoxicated at the time of injury [1, 3, 4, 8, 9, 10–11]. A major cause of enhanced morbidity and mortality in intoxicated patients with burns is the increased incidence of bacterial infections. Another potential complication in these patients is their inability to mount an appropriate host response to invading pathogens [3, 4, 12, 13, 14, 15–16]. Both clinical and experimental evidence suggest that burn injury results in suppressed immune effector responses [3, 4, 12, 14, 15–16]. We have shown that ethanol intoxication exacerbates the suppression of intestinal T cell function after burn injury, and this decline leads to increased gut bacterial translocation. We and others have further demonstrated that mice receiving a combined insult of ethanol and burn injury exhibit impaired delayed type hypersensitivity, T cell proliferation, and IL‐2 production and enhanced susceptibility to infection [12, 17, 18, 19, 20, 21, 22–23]. Additional findings from our laboratory have shown a decrease in Th1 and Th17 effector functions in rodent models of ethanol and burn injury [12, 17, 18, 19, 20, 21, 22, 23, 24–25]. Together, these studies suggest that a decrease in T cell effector functions contributes to increased infection after ethanol and burn injury.

T cell‐mediated immunity plays a major role in host defense against a variety of pathogens. Under normal physiologic conditions, the differentiation of naive T cells into its different subsets depends on a variety of intra‐ and extracellular factors. The initial stimulation of T cells with Ag results in a cascade of intracellular signaling including the activation of Src‐kinases and MAPKs. The presence of extracellular factors further helps these activated T cells to differentiate into various subsets. For example, the differentiation of naive T cells into Th17 cells depends on the presence of TGF‐β1 and IL‐6 [26, 27]. Studies have shown that TGF‐β1 and IL‐6 initiate differentiation and that IL‐23 expands and stabilizes Th17 cells. Binding of IL‐23 to its receptor complex activates multiple STAT proteins leading to the expression of hallmark Th17 transcription factors: retinoic acid‐related orphan receptor (ROR)‐γt and aryl hydrocarbon receptor (AHR) [26, 28]. Although both ROR‐γt and AHR are significant in the production of Th17 effector cytokines IL‐17 and ‐22, several lines of evidence indicate that AHR predominately initiates the release of IL‐22 [25, 28, 29]. Once released, Th17 effector cytokines, particularly IL‐22, have been proposed to regulate mucosal barriers, including the gut and lung epithelial lining, where they help maintain an intact physical barrier and induce expression of antimicrobial peptides to prevent any microbial bacterial invasion. A recent study from our laboratory demonstrated that ethanol intoxication before burn injury suppresses Peyer's patch (PP) Th17 cell responses [24, 25]. In this study, we expanded our analysis to mesenteric lymph nodes (MLNs) and spleen to determine whether the Th17 suppression is global or is specific to PPs. To delineate the mechanism underlying suppressed Th17 effector functions, we assessed the role of IL‐6, IL‐23, and TGF‐β. Experiments were performed to examine whether ethanol consumption combined with burn injury (hereinafter referred to as ethanol and burn injury or burn ethanol injury) influences the expression of AHR and whether the presence of IL‐23 modulates AHR expression in T cells after ethanol and burn injury. Finally, using the AHR agonist, 6‐formylindolo (3, 2‐b) carbazole (FICZ), we assessed whether the AHR ligand FICZ modulates the Th17 cytokine production after ethanol and burn injury. Our results suggest that IL‐23 alone is sufficient to restore Th17 cytokines after ethanol and burn injury and that IL‐6 and TGF‐β do not influence IL‐23‐mediated release of Th17 effector cytokines.

MATERIALS AND METHODS

Animals and reagents

Male C57/BL6 mice (22–25 g) were obtained from Charles River Laboratories (Wilmington, MA, USA). IFN‐γ and IL‐6 ELISA kits were obtained from BD Biosciences (San Diego, CA, USA); IL‐2, IL‐22, and TGF‐β ELISA kits and recombinant mouse (r)IL‐23 from eBioscience (San Diego, CA, USA); IL‐17 ELISA kit, mouse rTGF‐β, and mouse rIL‐6, anti‐mouse IL‐6 Ab, and anti‐mouse TGF‐β Ab from R&D Systems (Minneapolis, MN, USA); primers to AHR, CYP1A1, and β‐actin and the MirVana miRNA Isolation Kit, High Capacity cDNA Reverse Transcription Kit, and TaqMan Gene Expression Master Mix from Thermo Fisher Scientific (Waltham, MA, USA); and the AHR agonist FICZ from Abcam (Cambridge, MA, USA).

Mouse model of acute ethanol intoxication and burn injury

As described elsewhere [22, 23], 22–25 g male mice were randomly divided into 4 groups: sham vehicle, sham ethanol, burn vehicle, and burn ethanol. In the ethanol group, mice were gavaged with 0.4 ml of 25% ethanol (∼2.9 g/kg) and in the sham vehicle group, with 0.4 ml water. Four hours after the gavage, a time when blood ethanol levels were in the range of 90–100 mg/dl in the ethanol‐treated mice, the mice were anesthetized with a mixture of ketamine and xylazine by i.p. injection and transferred into a template fabricated to expose ∼12.5% of the total body surface area (TBSA). For burn injury, mice were immersed in ∼90°C water bath for ∼7 s, dried immediately, and resuscitated with 1.0 ml physiologic saline by i.p. injection. After recovery from anesthesia, the mice were returned to their cages and allowed food and water ad libitum. All the animal procedures were performed in compliance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD, USA) and were approved by the Loyola University Chicago Health Sciences Division Institutional Animal Care and Use Committee.

Preparation of MLN, PP, and spleen cell suspension

On days 1, 3, and 7 after injury, mice were euthanized, and the abdominal cavity exposed via midline incision. MLNs, PPs, and spleen were collected aseptically. To prepare single‐cell suspensions, MLNs were gently crushed in HBSS solution (Thermo Fisher Scientific) supplemented with 10 mM HEPES, 50 µg/ml gentamicin, and 100 U/ml penicillin with 100 µg/ml streptomycin (complete HBSS). Cell suspensions were centrifuged at 290 g for 15 min at 10°C. Supernatants were discarded, and cells were washed with 5 ml RPMI‐1640 supplemented with 2 mM l‐glutamine, 10 mM HEPES, 50 µg/ml gentamicin, 100 U/ml penicillin and 100 µg/ml streptomycin, and 10% FCS (complete RPMI‐1640) and were resuspended in complete RPMI‐1640.

To isolate the PP cells, the PPs were incubated in complete HBSS containing 0.5 mg/ml collagenase D, for 15 min at 37°C [25]. After collagenase D treatment, PPs were crushed to prepare single‐cell suspensions, filtered through a 70 μm nylon filter, washed, and resuspended in complete RPMI‐1640.

To prepare spleen single‐cell suspensions, spleens were gently crushed in complete HBSS. Cell suspensions were centrifuged at 290 g for 15 min at 10°C. The RBCs were lysed by adding 9 ml of sterile distilled H2O followed by 1 ml of 10× PBS and centrifuged at 290 g for 15 min at 10°C. Supernatant was discarded, and the cells were washed and resuspended in complete RPMI‐1640 for cell culture. To further enrich the T cell population, 106–107 total spleen cells were resuspended in 90 µl of separation buffer (PBS containing 0.5% BSA and 2 mM EDTA) and incubated with 10 µl of CD90 (Thy1.2) MicroBeads (Miltenyi Biotec, Auburn, CA, USA) for 15 min at 4°C. The cells were washed with separation buffer and run through separation columns (Miltenyi Biotec) in a magnetic field. Purified T cells were obtained by flushing out magnetically labeled cells from the separation columns [22, 23].

Measurement of cytokines

As described [23, 25], the cells isolated from MLN, PPs, and spleen (5 × 105 cells/well) were cultured in complete RPMI‐1640 in 96‐well plates precoated with anti‐CD3 Ab (5 µg/ml) in the presence of anti‐CD28 Ab (1 µg/ml) at 37°C and 5% CO2 for 48 h. In some experiments, these cells were cultured with anti‐CD3 and ‐CD28 Abs in the presence or absence of rIL‐6 (20 ng/ml), rTGF‐β (5 ng/ml), rIL‐23 (10 ng/ml), anti‐mouse IL‐6 Ab (1 or 10 µg/ml), and anti‐mouse TGF‐β Ab (1 or 10 µg/ml) for 48 h [30, 31]. The supernatants were harvested to test IL‐2, IFN‐γ, IL‐22, IL‐17, IL‐6, and TGF‐β levels by using ELISA kits according to the manufacturer's instructions. In separate experiments, spleen T cells were cultured with anti‐CD3 and ‐CD28 Abs in the presence or absence of AHR agonist FICZ at 200 nM for 48 h. The supernatants were harvested to determine IL17, IL‐22, and IFN‐γ.

Measurement of spleen T cell AHR and CYP1A1 mRNA expression

Spleen T cells (5 × 105 cells/well) were cultured in complete RPMI‐1640 in 96‐well plates in the presence or absence of anti‐CD3, anti‐CD28 Abs, rIL‐23, and FICZ at 37°C and 5% CO2 for 16 or 48 h. The cells were collected for isolation of total RNA by using the mirVana miRNA Isolation Kit according to the manufacturer's instructions. The total RNA concentration was determined by using a Nanodrop spectrophotometer (Thermo Fisher Scientific). The total RNA (1 μg) was used for cDNA reverse transcription by using a High Capacity cDNA Reverse Transcription Kit, according to the manufacturer's instructions. The expressions of AHR and CYP1A1 were analyzed by RT‐PCR with respective primers and normalized with β‐actin.

Statistical analysis

The data are presented as means ± sem and were analyzed by using 1‐way ANOVA with Tukey‐Kramer multiple‐comparisons test or Student's 2‐tailed t test (In‐Stat; GraphPad Software Inc., La Jolla, CA, USA). P < 0.05 indicates statistical significance.

RESULTS

Th1 and Th17 cell responses in MLN, PP, and spleen

There were no significant differences in IL‐17 (Fig. 1A), IL‐22 (Fig. 1B), IL‐2 (Fig. 1C), or IFN‐γ (Fig. 1D) in cells isolated from MLNs, PPs, and spleens in sham ethanol– and sham vehicle–treated mice. Furthermore, there was no change in IL‐17, IL‐22, IL‐2, and IFN‐γ levels in cells isolated from all 3 organs in the burn vehicle–treated mice compared with the sham vehicle–treated mice except for PP IL‐22 and spleen IFN‐γ. However, as reported earlier [22, 23, 25], significant decreases in IL‐17, IL‐22, IL‐2, and IFN‐γ were observed in cells of all 3 lymphoid organs after a combined insult of ethanol and burn injury compared with sham vehicle–treated mice except MLN IL‐22. In addition, as compared with burn vehicle, the burn ethanol group exhibited a significant decrease in the release of IL‐17 by MLNs and PPs; IL‐22 release by PPs and spleen; IL‐2 release by MLNs and PPs; and IFN‐γ release by MLNs and PPs.

Figure 1.

Figure 1

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Th1 and Th17 responses 1 d after combined ethanol and burn injury.

At 1 d after injury, mice were euthanized, and MLNs, PPs, and spleens were collected for cell isolation. Total cells (5 × 105 cells/well) were cultured with plate‐bound anti‐CD3 (5 µg/ml) and anti‐CD28 (1 µg/ml) Abs for 48 h, and supernatants were collected to determine IL‐17 (A), IL‐22 (B), IL‐2 (C), and IFN‐γ (D). Data are means ± sem from 5 to 9 animals per treatment group (n = 5, sham vehicle; 6, sham ethanol; 7, burn vehicle; and 9, burn ethanol). *P < 0.05 vs. sham vehicle by one‐way ANOVA with the Tukey‐Kramer multiple‐comparisons test; # P < 0.05 vs. sham vehicle by t test.

Next, we assessed whether suppression of Th1 and Th17 responses was time dependent. To accomplish this, mice were euthanized on day 3 after ethanol and burn injury and Th1 and Th17 effector functions were determined. Similar to day 1, there were no significant differences in IL‐17 ( Fig. 2A ), IL‐22 (Fig. 2B), IL‐2 (Fig. 2C), and IFN‐γ (Fig. 2D) levels in MLN, PP, and spleen of sham ethanol– and sham vehicle–treated mice. In the burn vehicle–treated mice, these cytokine responses in cells obtained from all 3 organs trended toward a decrease. IL‐17 in MLNs and PPs, IL‐2 in MLNs, and IFN‐γ in MLNs and spleen were significantly decreased in burn vehicle–treated mice compared with sham vehicle–treated mice. However, similar to 1 d after injury, the release of IL‐17, IL‐22, IL‐2, and IFN‐γ by cells of all 3 lymphoid organs continue to significantly decrease in burn ethanol mice compared with sham vehicle–treated mice. Furthermore, except splenic IFN‐γ, the levels of other Th1 and Th17 cytokines were found to be significantly lower in cells from mice undergoing a combined insult of ethanol and burn injury compared with the burn vehicle group.

Figure 2.

Figure 2

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Th1 and Th17 responses on day 3 after combined ethanol and burn injury.

Three days after injury, mice were euthanized, and MLNs, PPs, and spleens were collected for cell isolation. Total cells (5 × 105 cells/well) were cultured with plate‐bound anti‐CD3 (5 µg/ml) and anti‐CD28 (1 µg/ml) Abs for 48 h, and supernatants were collected to determine IL‐17 (A), IL‐22 (B), IL‐2 (C), and IFN‐γ (D). Data are means ± sem from 3 to 7 animals per treatment group (n = 4, sham vehicle; 3, sham ethanol; 7, burn vehicle; and 7, burn ethanol). *P < 0.05 vs. sham vehicle by one‐way ANOVA with Tukey‐Kramer multiple comparisons test; # P < 0.05 vs. sham vehicle by t test.

The results presented in Figs. 1 and 2 clearly show that the amplitude of Th1 and Th17 cytokine responses in various lymphoid organs are different, but the trend of a decrease in T cell cytokines after ethanol and burn injury remains the same. Although these differences could be related to the inherent tendency of cells in each specific lymphoid organ, the number of CD3+ cells in each lymphoid organ were not found to be significantly different between the sham vehicle and burn ethanol groups (unpublished results).

To further examine the time course of the dysregulated Th1 and Th17 effector function, we assessed Th1 and Th17 responses on day 7 after ethanol and burn injury. In this experiment, only spleen cells were used, as the Th1 and Th17 responses in all 3 lymphoid organs followed a similar trend 1 and 3 d after ethanol and burn injury. The results summarized in Table 1 clearly show no significant differences in any Th1 and Th17 cytokine produced by cells harvested from any experiment group. These results suggest that Th1 and Th17 responses are normalized by day 7 after ethanol and burn injury.

Table 1.

Th1 and Th17 responses on day 7 after combined ethanol and burn injury

Sham vehicle Burn vehicle Burn ethanol
IL‐17 (pg/ml) 834.87 ± 122.86 861.48 ± 232.63 844.53 ± 268.67
IL‐22 (pg/ml) 158.16 ± 6.33 132.63 ± 13.03 131.46 ± 9.56
IL‐2 (pg/ml) 249.30 ± 23.50 206.87 ± 28.18 195.45 ± 26.66
IFN‐γ (ng/ml) 57.29 ± 6.53 44.44 ± 6.30 57.37 ± 8.20
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Seven days after injury, mice were euthanized, and spleens were collected. Total spleen cells (5 × 105 cells/well) were cultured with plate‐bound anti‐CD3 (5 µg/ml) and anti‐CD28 (1 µg/ml) Abs for 48 h, and supernatants were collected to determine IL‐17, IL‐22, IL‐2, and IFN‐γ. Data are means ± sem from 5 to 7 animals per group (n = 5, sham vehicle; 7, burn vehicle; and 6, burn ethanol).

Subsequent studies were performed with cells from the spleen. The reason for selecting the spleen in subsequent studies is the availability of enough cells to carry out experiments in various culture conditions. To reduce the variability, we wanted to compare the cytokine responses from the same mouse in various treatment conditions. The number of cells from MLNs and PPs was not sufficient for all the treatment groups, and they were not used for these studies. In addition, because burn ethanol mice are the only group that showed maximum suppression of Th1 and Th17 responses 1 and 3 d after injury, only 2 experimental groups—sham vehicle–treated and burn ethanol—were used to delineate the mechanism of suppressed Th17 effector function after ethanol and burn injury.

Assessment of IL‐6, TGF‐β, and IL‐23 production by splenocytes

As mentioned previously, IL‐6 and TGF‐β initiates the Th17 cell differentiation, but their expansion and function is dependent on IL‐23 [26, 27]. IL‐23 is composed of an IL‐12p40 subunit that is shared with IL‐12 and the IL‐23p19 subunit. In a study from our laboratory, we have shown that ethanol combined with burn injury suppresses IL12/23p40 and IL‐23p19 in PP cells [25]. In the current experiment, we determined whether ethanol combined with burn injury also influences IL‐6 and TGF‐β levels. Spleen cells were cultured with plate‐bound anti‐CD3 and soluble anti‐CD28 Abs or different doses of LPS (100 ng/ml or 1 µg/ml) for 24 h. We observed a demonstrable decrease in IL‐6 levels in all three conditions after ethanol and burn injury, compared with sham vehicle–treated animals ( Fig. 3A ). However, spleen cells cultured with anti‐CD3 and anti‐CD28 Abs produced several‐fold higher IL‐6 in both sham vehicle–treated and burn ethanol mice compared with cells cultured with LPS. Similar to IL‐6, there was a significant decrease in TGF‐β levels in spleen cells cultured with anti‐CD3 and ‐CD28 in burn ethanol animals compared with sham vehicle–treated animals (Fig. 3B). TGF‐β levels were undetectable in cells cultured with LPS in both sham vehicle–treated animals and burn ethanol animals. IL‐23 in splenocytes was also undetectable in all groups. Although TGF‐β and IL‐6 are produced by many cell types, the findings of higher levels of IL‐6 and TGF‐β in cells stimulated with anti‐CD3/anti‐CD28 Abs suggest that T cells are likely the major source for these cytokines in the splenic compartment.

Figure 3.

Figure 3

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IL‐6 and TGF‐β levels after combined ethanol and burn injury.

One day after injury, mice were euthanized and spleens were collected for cell isolation. Total cells (5 × 105 cells/well) were cultured with plate‐bound anti‐CD3 (5 µg/ml) and anti‐CD28 (1 µg/ml) Abs or LPS (100 ng/ml or 1 µg/ml) for 24 h, and supernatants were collected to determine IL‐6 (A) and TGF‐β (B). Data are means ± sem from 5 to 6 animals per treatment group (n = 5, sham vehicle, and 6, burn ethanol). *P < 0.05 vs. respective sham vehicle group, by t test.

IL‐23 restoration of Th17 cell effector cytokines is not dependent on the presence of IL‐6 and TGF‐β after ethanol and burn injury

To further delineate the role of IL‐23, IL‐6, and TGF‐β in suppressed Th17 effector cytokines, we determined whether ex vivo reconstitution of IL‐6, TGF‐β, and IL‐23 in cultured splenic cells would normalize Th17 effector cytokines after ethanol combined with burn injury. Isolated spleen cells were cultured with plate‐bound anti‐CD3 and soluble anti‐CD28 Abs in the presence and absence of rIL‐6 (20 ng/ml) and rTGF‐β (5 ng/ml) combined, with or without rIL‐23 (10 ng/ml) for 48 h. Supernatants were collected to determine IL‐17 and IL‐22 levels. Results presented in Fig. 4 clearly show that addition of rIL‐6 and rTGF‐β in the culture medium did not restore IL‐17, IL‐22, IL‐2, or IFN‐γ production after ethanol and burn injury. However, the reconstitution of IL‐23 in culture medium increased IL‐17 2‐fold higher than without rIL‐23 in both sham vehicle–treated and burn ethanol mice. Furthermore, IL‐23 increased IL‐22 levels 5‐fold in the sham vehicle–treated group and 22‐fold in the burn ethanol mice compared with their respective untreated group of mice. Yet, the restoration of IL‐23, similar to that of IL‐6 and TGF‐β, did not influence IL‐2 and IFN‐γ in both the sham vehicle–treated and burn ethanol animals. The restoration of IL‐23 in culture medium increased IL‐6 ( Fig. 5A ) in both the sham vehicle–treated and burn ethanol, but it did not influence TGF‐β levels (Fig. 5B). Then, we determined whether the IL‐23 restoration of Th17 responses is dependent on IL‐6 and TGF‐β. Spleen cells were cultured with anti‐CD3 and ‐CD28 Abs plus rIL‐23 (10 ng/ml) in the presence of an anti‐IL‐6 (1 or 10 μg/ml) and anti‐TGF‐β (1 or 10 μg/ml) Abs cocktail, to neutralize IL‐6 and TGF‐β. Supernatants from these cultures were harvested and analyzed for IL‐17 and IL‐22 levels. Figure 5 clearly shows that the levels of IL‐6 and TGF‐ β were completely neutralized in cells cultured with anti‐IL‐6 and TGF‐β Abs, but this neutralization did not influence the IL‐23‐mediated release of IL‐17 and ‐22 in both sham vehicle and burn ethanol group. Together, these results suggest that IL‐23 alone is sufficient to restore Th17 effector cytokines after ethanol and burn injury.

Figure 4.

Figure 4

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Effect of rIL‐6, rTGF‐β, and rIL‐23 on Th1 and Th17 responses after combined ethanol and burn injury.

One day after injury, mice were euthanized, and spleens were collected for cell isolation. Total cells (5 × 105 cells/well) were cultured with plate‐bound anti‐CD3 (5 µg/ml) and anti‐CD28 (1 µg/ml) Abs in the presence or absence of rIL‐6 (20 ng/ml), rTGF‐β (5 ng/ml), and rIL‐23 (10 ng/ml) for 48 h and supernatants were collected to determine IL‐17 (A), IL‐22 (B), IL‐2 (C), and IFN‐γ (D). The experiment was conducted using splenocytes from the animals used in Fig. 1. Data are means ± sem from 5 to 9 animals per group (n = 5, sham vehicle, and 9, burn ethanol). *P < 0.05 vs. all except CD3 CD28 IL‐6– and TGF‐β–treated groups, by one‐way ANOVA with the Tukey‐Kramer multiple‐comparisons test. # P < 0.05 vs. respective other groups, except CD3 CD28 IL‐6– and TGF‐β–treated groups, by t test.

Figure 5.

Figure 5

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IL‐23‐mediated Th17 cytokine restoration is not dependent on IL‐6 and TGF‐β after combined ethanol and burn injury.

One day after injury, mice were euthanized, and spleens were collected. Total spleen cells (5 × 105 cells/well) were cultured for 48 h with plate‐bound anti‐CD3 (5 µg/ml) and anti‐CD28 (1 µg/ml) Abs, in the presence or absence of rIL‐23 (10 ng/ml), anti‐IL‐6 Ab (1 or 10 µg/ml), anti‐TGF‐β Ab (1 or 10 µg/ml), mouse IgG (10 µg/ml), and rat IgG (10 µg/ml), and supernatants were collected to determine IL‐6 (A), TGF‐β (B), IL‐17 (C), and IL‐22 (D). Data are means ± sem from 5 to 7 animals per group (n = 5, sham vehicle; and 7, burn ethanol). *P < 0.05 vs. other groups. # P < 0.05 vs. other groups except cells cultured with CD3 CD28 plus rIL‐23. @ P < 0.05 vs. other sham vehicle groups.

AHR is essential for IL‐23 restoration of Th17 responses after ethanol and burn injury

AHR is a ligand‐dependent transcription factor that is involved in Th17 cell proliferation and differentiation [26, 28]. We first examined whether ethanol and burn injury influences AHR expression in T cells and whether rIL‐23 modulates AHR expression in these cells. Splenic T cells were isolated and cultured with plate‐bound anti‐CD3 and soluble anti‐CD28 Abs, in the presence or absence of rIL‐23 for 16 h. Cells were collected, and mRNA was isolated. Expression of AHR was determined by RT‐PCR. There was a significant decrease in AHR mRNA expression in T cells cultured with anti‐CD3 and ‐CD28 in burn ethanol animals compared with sham vehicle–treated animals ( Fig. 6 ). However, T cells cultured with anti‐CD3 and ‐CD28 Abs plus rIL‐23 attenuated the decrease in AHR expression after ethanol and burn injury. This finding suggests that ethanol and burn injury decreases AHR expression, which can lead to a decrease in Th17 effector cytokines.

Figure 6.

Figure 6

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Effect of rIL‐23 on T cell AHR mRNA expression after combined ethanol and burn injury.

One day after injury, mice were euthanized, and spleens were collected for T cell isolation. T cells (5 × 105 cells/well) were cultured with plate‐bound anti‐CD3 (5 µg/ml) and anti‐CD28 (1 µg/ml) Abs in the presence or absence of rIL‐23 (10 ng/ml) for 16 h, cells were collected, and mRNA was isolated. Expression of AHR mRNA was determined by RT‐PCR and normalized by β‐actin. Data are means ± sem (n = 7 animals per group). *P < 0.05 vs. other groups.

The AHR agonist FICZ enhanced IL‐22, but not IL‐17 and IFN‐γ

FICZ is a known AHR ligand that has been used in studies to induce IL‐22 release [29, 32]. We examined whether direct stimulation of AHR with FICZ influences the release of Th17 cytokines after ethanol and burn injury. T cells were cultured with anti‐CD3 and ‐CD28 Abs, in the presence or absence of 200 nM FICZ for 48 h. This dose was selected from published studies [29, 32]. The supernatants were collected and measured for IL‐17, IL‐22, and IFN‐γ. T cells cultured with anti‐CD3 and ‐CD28 Abs plus 200 nM FICZ demonstrably increased IL‐22 production compared with T cells cultured with anti‐CD3 and ‐CD28 Abs without Abs FICZ in both the sham vehicle–treated and burn ethanol groups (Fig. 7B). T cells cultured with anti‐CD3 and ‐CD28 Abs plus FICZ did not increase levels of IL‐17 (Fig. 7A) and IFN‐γ (Fig. 7C) release compared with T cells cultured with anti‐CD3 and ‐CD28 Abs alone in both sham vehicle and burn ethanol animals. Next, we determined the expression of CYP1A1, which is transcriptionally regulated by AHR. There was a decrease in CYP1A1 expression in T cells cultured with anti‐CD3 and ‐CD28 Abs in burn ethanol compared with sham vehicle animals ( Fig. 8 ). However, T cells cultured with anti‐CD3 and ‐CD28 Abs plus 200 nM FICZ had an ∼20‐fold increase in CYP1A1 expression compared with T cells cultured with anti‐CD3 and ‐CD28 Abs alone in sham vehicle–treated and burn ethanol animals.

Figure 7.

Figure 7

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Effect of FICZ in Th1 and Th17 responses after combined ethanol and burn injury.

One day after injury, mice were euthanized, and spleens were collected for T cell isolation. T cells (5 × 105 cells/well) were cultured with plate‐bound anti‐CD3 (5 µg/ml) and anti‐CD28 (1 µg/ml) Abs in the presence or absence of FICZ (200 nM) for 48 h, and supernatants were collected to determine IL‐17 (A), IL‐22 (B), and IFN‐γ (C). Data are means ± sem from 5 to 6 animals per group (n = 5, sham vehicle, and 6, burn ethanol). *P < 0.05 vs. other groups. # P < 0.05 vs. respective sham vehicle group.

Figure 8.

Figure 8

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Effect of FICZ on CYP1A1 mRNA expression after combined ethanol and burn injury.

One day after injury, mice were euthanized, and spleens were collected for T cell isolation. T cells (5 × 105 cells/well) were cultured with plate‐bound anti‐CD3 (5 µg/ml) and anti‐CD28 (1 µg/ml) Abs in the presence or absence of FICZ (200 nM) for 48 h and were collected and the RNA isolated. Expression of CYP1A1 mRNA was determined by RT‐PCR and normalized by β‐actin; P = 0.0212. Data are means ± sem from 4 to 6 animals per group (n = 4, sham vehicle, and 6, burn ethanol). *P < 0.05 vs. other groups.

DISCUSSION

Our findings clearly demonstrate that acute ethanol intoxication combined with burn injury significantly decreased Th1 and Th17 effector cytokines in MLN, PP, and spleen cells, 1 and 3 d after injury. These data add to our earlier observations [22, 23, 25] suggesting that ethanol intoxication combined with burn injury globally suppresses Th1 and Th17 responses on days 1 and 3 after injury. Both Th1 and Th17 responses were normalized by day 7 after combined injury. These findings suggest that a decrease in T cell effector functions within 24 h after injury can contribute to a decrease in host defense and increased susceptibility to infection after alcohol and burn injury. The levels of IL‐2, IFN‐γ, IL‐22, and IL‐17 in cells isolated from mice after burn injury alone trended toward a decrease, but these decreases were not found to be significant when compared with sham‐injured mice, in contrast to several studies suggesting T cell suppression after burn injury [33, 34–35]. A potential cause of this difference is that some researchers have used 25% or more TBSA burns compared with the 12.5% TBSA used in our study.

Th17 differentiation is a multistep process and is dependent on several cytokines. Studies have shown that a combination of TGF‐β and IL‐6 is necessary for Th17 cell differentiation [26, 27]. IL‐23, a member of the IL‐12 family, is another cytokine released by APCs (e.g., dendritic cells and Mϕs) that further expands and stabilizes Th17 cells. IL‐23 binds to its receptor and activates the JAK/STAT pathway, leading to activation of multiple STAT proteins [36, 37–38]. The phosphorylation of STAT3 leads to activation of ROR‐γt and AHR. AHR has been suggested to be the major transcription factor involved in the release of IL‐22 [28, 29, 32, 39]. In addition, RORα and interferon regulatory factor (IRF)‐4 are shown to play important roles in the production of the Th17 effector cytokines IL‐17 and ‐22 [40, 41]. Because IL‐6, TGF‐β, and IL‐23 are necessary for the differentiation and proliferation of Th17 cells, we determined whether ethanol and burn injury influences the release of IL‐6, TGF‐β, and IL‐23 and thereby contributes to decreases in Th17 effector cytokines. We observed a significant decrease in IL‐6 and TGF‐β in splenic cells harvested from mice undergoing ethanol and burn injury compared with sham vehicle–treated mice. Similarly, a decrease in IL‐23 was observed in cells from PPs after ethanol combined with burn injury, compared with the sham vehicle animals in our published study [25]. To further understand the roles of IL‐6, TGF‐β, and IL‐23 in suppression of IL‐17 and ‐22, we performed ex vivo experiments in which splenic cells were cultured in the presence or absence of rIL‐6 plus rTGF‐β, with or without rIL‐23. Results from these experiments (Fig. 4), clearly demonstrate that IL‐23 plays a major role in restoration of the Th17 effector cytokines IL‐17 and ‐22 after ethanol and burn injury, and the effect is not dependent on IL‐6 and TGF‐β. Although our results are in contrast to those suggesting that IL‐6 and TGF‐β are sufficient to induce naive T cell differentiation into Th17 cells [27, 42, 43], our findings clearly support others suggesting that IL‐23 is a major player in Th17 release of IL‐17 and IL‐22. IL‐23 strongly upregulates the expression of IL‐17A, IL‐17F, IL‐22, and CCL20 in human T cells [44, 45]. IL‐23‐deficient mice have lower frequencies of Th17 cells [46, 47]. Consistent with these findings, we observed that IL‐23 enhanced IL‐22 and ‐17 by activating AHR transcription factor after ethanol combined with burn injury. In another study from our laboratory, we showed that PP cells cultured with an AHR inhibitor, CH‐223191, before IL‐23 treatment inhibits the IL‐23‐mediated IL‐22 production after ethanol and burn injury [25]. Together, these data suggest a role of AHR in IL‐23‐mediated Th17 effector cytokine production after ethanol and burn injury.

AHR has multiple downstream target genes, including the cytochrome p450 family, CYP1A1, ‐A2, and ‐B1, through which it recruits other cofactors to mediate its effector responses [48]. T cells from AHR‐deficient mice fail to produce IL‐22 [29]. Knockdown of AHR via AHR siRNA in human memory T cells downregulates CYP1A1 mRNA expression and inhibits IL‐22 release. Treatment of polarized Th17 cells with AHR agonists, βNF or FICZ induces IL‐22 production [49]. In this study, we observed that T cells cultured with anti‐CD3 plus anti‐CD28 Abs in the presence of FICZ significantly increased IL‐22 production and promoted CYP1A1 mRNA expression in both sham vehicle and burn ethanol mice. However, FICZ did not influence IL‐17 and IFN‐γ production. Trifari et al. [49] demonstrated that IL‐22‐producing human T cells coexpress CCR6, ‐4, and ‐10, a finding that is distinct from both Th17 and Th1 cells. Knockdown of AHR in CD4+ T cells inhibited only IL‐22 production, but did not influence production of IL‐17 and IFN‐γ. The expression of ROR type C (RORC) was associated with IL‐17 production. The AHR agonists βNF and FICZ promoted expression of CYP1A1 and IL‐22 production, but not expression of RORC and production of IL‐17 and IFN‐γ in human T cells. However, AHR agonists had opposing effects in IL‐17‐producing cells in human and mouse. Treatment of mouse Th17 cells with FICZ, not only increased IL‐22‐producing cells, but also increased IL‐17‐producing cells. In contrast, we observed that treatment of T cells with FICZ increased IL‐22, but not IL‐17, in T cells. Together, these findings suggest that regulation of IL‐17 production by AHR is dependent on the species and T cell types.

In conclusion, our findings indicate that acute ethanol intoxication combined with burn injury suppressed Th1 and Th17 responses, not only in intestinal lymphoid organs, but also in systemic lymphoid organs 1 and 3 d after injury. However, Th1 and Th17 responses are restored by day 7. Our findings also showed that IL‐23 played a critical role in restoration of IL‐17 and ‐22 by activation of the AHR pathway after ethanol combined with burn injury. Treatment of T cells with the AHR agonist FICZ enhanced IL‐22 production, but not IL‐17 after injury. IL‐22 plays a major role in maintaining gut immunity and barrier integrity and regulates intestinal inflammation, tissue repair, and expression of antimicrobial peptides [50]. IL‐22 knockout mice with Citrobacter rodentium infection experience increased mucosal hyperplasia, submucosal inflammation, bacteria infiltration, and mortality compared with wild‐type mice with C. rodentium infection [51]. In a previous study, we have shown that treatment of mice with rIL‐22 normalized the expression of the antimicrobial peptides Reg3β and ‐3γ and attenuated the increase in intestinal permeability after ethanol combined with burn injury [24]. These findings suggest that AHR agonists, such as FICZ and βNF, may provide innovative therapeutic agents to maintain the intestinal barrier after injury, which may be useful in the treatment of patients under the influence of ethanol who sustain a burn injury.

AUTHORSHIP

X.L. and M.A.C. conceived the work. X.L., A.R.C., A.M.H., N.L.M., and M.A.C. designed the experiments. X.L., A.R.C., A.M.H., and N.L.M., performed the experiments. X.L., A.R.C., A.M.H., N.L.M., and M.A.C. wrote the manuscript.

DISCLOSURES

The authors declare no conflicts of interest.

ACKNOWLEDGMENTS

This study was supported by U.S. National Institutes of Health, National Institute on Alcohol Abuse and Alcoholism Grants R01 AA015731, T32 AA013527, and F31 AA024367 (to A.M.H).

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