The ACTH test fails to correctly identify adrenal insufficiency in early/acute and middle/subacute stages of sepsis. First, we performed the ACTH test in wild-type (C57BL/6J) mice under physiological conditions. The mice exhibited a normal adrenal stress response to ACTH (0.1 IU) stimulation, as indicated by stress levels of corticosterone in circulation (Figure 1A) and a ΔGC of greater than 90 ng/mL (Figure 1B). Upon CLP challenge, sepsis induced stress levels of iGCs in circulation at the early/acute stage of sepsis (3 hours after CLP), with iGC levels gradually decreasing at 24 and 48 hours after CLP (Figure 1C). The mice displaced well-controlled inflammatory responses, as shown by high levels of interleukin-6 (IL-6) at the early/acute stage of sepsis (3 hours after CLP), with IL-6 levels under control at 24 and 48 hours after CLP (Figure 1C). Thus, the wild-type mice had a normal adrenal stress response and inflammatory response in sepsis. However, when we conducted the ACTH test in septic mice, the results were intriguing. At the early/acute stage of sepsis (3 hours after CLP), ACTH stimulation failed to induce additional GC production (Figure 1D), and all the septic mice had a ΔGC of less than 90 ng/mL (Figure 1E). Thus, all the septic mice were identified as having RAI or CIRCI by the ACTH test, even though the mice had a normal adrenal stress response to sepsis. At the middle/subacute stage of sepsis (24 hours aftert CLP), ACTH stimulation moderately increased GC levels (Figure 1D), but half of the mice were still identified as having RAI or CIRCI by the ACTH test (Figure 1E). At the late stage of sepsis (48 hours after CLP), ACTH stimulation induced a significant increase in corticosterone levels (Figure 1D), and most mice had a ΔGC of greater than 90 ng/mL (Figure 1E), thus being diagnosed with a normal adrenal stress response. It is worth noting that the wild-type mice had a normal adrenal stress response at all stages of sepsis, with no “insufficient GC relative to an increased demand” or “inadequate cellular corticosteroid activity for the severity of the patient’s critical illness.” Therefore, the ΔGC detected by the ACTH test is inappropriate for the diagnosis of adrenal insufficiency under septic conditions and may incorrectly diagnose RAI or CIRCI. Of note, the GC levels stimulated by ACTH (0.1 IU) were lower than those stimulated by CLP. Given that the clinical ACTH test dose is a super-stressor dose, we also performed the ACTH test with a super-stressor dose of ACTH (4 IU). The wild-type mice did not produce more iGCs at the acute stage of sepsis (3 hours after CLP) (Figure 1, F and G).

ACTH test fails to correctly identify adrenal stress response in early and middle stages of sepsis. (A and B) C57BL/6J mice were treated with 0.1 IU ACTH via subcutaneous injection. Serum corticosterone (A) and Δcorticosterone (B) levels were measured before and 1 hour after the ACTH test (n = 6). Data are presented as mean ± SEM. Statistical testing using 2-tailed unpaired Student’s t test. (C) C57BL/6J mice were challenged with CLP (25G, full ligation) for different times (0, 3, 24, 48 hours). Serum corticosterone and IL-6 levels were measured (n = 6–9). (D and E) C57BL/6J mice were challenged with CLP (25G, full ligation) for different times (0, 3, 24, 48 hours). Then, the mice were treated with 0.1 IU ACTH. Serum corticosterone (D) and Δcorticosterone (E) levels were measured before and 1 hour after the ACTH test (n = 6). (F and G) C57BL/6J mice were challenged with CLP for different times (0, 3, 24, 48 hours). Then, mice were treated with 4 IU ACTH. Serum corticosterone (F) and Δcorticosterone (G) levels were measured before and 1 hour after the ACTH test (n = 7–9). Data are presented as mean ± SEM. Statistical testing using 1-way ANOVA with Tukey’s multiple-comparison correction. (H and I) C57BL/6J mice were challenged with or without CLP for 3 hours (H) and stimulated with ACTH (4 IU) or PBS for 45 minutes (I). GC synthesis–related gene expression in the adrenal gland was analyzed by RNA-seq analysis. Data are presented as mean ± SEM. Statistics using 2-way ANOVA with Tukey’s multiple-comparison correction. NS, no significance; *P < 0.05; **P < 0.01; ****P < 0.0001. For RNA-seq analysis, pairwise comparisons between the various conditions were run using a negative binomial generalized log-linear model through the glmLRT fit function in edgeR (https://bioconductor.org/packages/release/bioc/html/edgeR.html).

To understand why the ACTH test cannot stimulate more iGC production at the acute stage of sepsis, we looked at ACTH-mediated steroidogenesis in the adrenal gland. As shown in Figure 1H, upon CLP challenge, the expression of melanocortin 2 receptor accessory protein (Mrap) was upregulated by 10-fold, indicating the trafficking of the ACTH receptor (MC2R) to the cell membrane in response to septic stress. The downstream cAMP-responsive element modulator (Crem) was also upregulated by 10-fold. CLP induced 10-, 2.6-, and 3-fold increases in SR-BI (Scarb1, uptake of cholesterol from HDL), LDL receptor (Ldlr, uptake of cholesterol from LDL), and HMG-CoA reductase expression (Hmgcr, de novo cholesterol synthesis), respectively. CLP induced 2- to 3-fold increases in Star and Cyp11b1 expression, the downstream key regulators of GC synthesis. Thus, sepsis induces robust ACTH-mediated steroidogenesis. However, all the above-listed key genes in GC synthesis were not further upregulated by the ACTH test (Figure 1I). Corticosterone is synthesized from cholesterol through a cascade of oxidative reactions that convert corticosterone precursors to corticosterone. These oxidative reactions are mediated by specific enzymes. We examined the expression of enzymes that convert corticosterone precursors to corticosterone. As shown in Figure 1I, the ACTH test did not induce more expression of the key enzymes, including Cyp11a1 (catalyzes the conversion of cholesterol to pregnenolone, the first step in steroidogenesis), Hsd3b1 (converts pregnenolone to progesterone), Cyp21a1 (converts progesterone to 11-deoxycorticosterone), and Cyp11b1 (converts 11-deoxycorticosterone to corticosterone, the final step in this pathway). This supports our conclusion that the ACTH test does not induce more activation of the hypothalamic-pituitary-adrenal (HPA) axis at the acute stage of sepsis.

We quantified ACTH levels at 3 and 24 hours after CLP (Supplemental Figure 2; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.187487DS1). The sepsis-induced endogenous ACTH levels remained similar at both the acute and subacute stages of sepsis, consistent with previous reports in septic mice. This suggests that GC levels are regulated not only by endogenous ACTH levels but also by other factors. Early studies showed that IL-6, along with proinflammatory cytokines like TNF-α and IL-1β, stimulates the HPA axis, leading to increased production of GCs (33).

We noticed that the post-ACTH corticosterone levels seemed to remain rather at the same magnitude during the 48-hour follow-up in CLP animals, albeit different T0 levels. This suggests that the GC production may be capped by the stress levels. At the acute stage, the adrenal glands are in utmost stressed conditions so that the ACTH test could not induce more GC production. At 24 and 48 hours after CLP, the endogenous stress levels were significantly decreased, evidenced by low corticosterone concentrations and low inflammatory cytokine concentrations. At this point, the adrenal glands are ready to be stimulated again. Our earlier study showed that SR-BI–mediated uptake of cholesterol into the adrenal gland is a key step in the adrenal stress response (iGC production) in response to stress (30). To test our speculation, we examined adrenal SR-BI expression by Western blotting at 0, 4, and 20 hours after CLP. As expected, adrenal SR-BI expression was at high levels at 4 and 20 hours after CLP (Supplemental Figure 1). Upon stimulation by the ACTH test, iGCs returned to stress levels at 24 hours after CLP, and even higher at 48 hours after CLP by the super-stressor dose of ACTH at 4 IU (Figure 1, D–G).

We also noticed that the ACTH test stimulated higher GC levels in septic conditions than in physiological conditions (Figure 1, D and F). This suggests that the ACTH test acts together with other stimulators in sepsis to stimulate more GC production. As discussed above, IL-6, along with proinflammatory cytokines like TNF-α and IL-1β, stimulates the HPA axis, leading to increased production of GCs (33).

In sum, we demonstrated that the ACTH test fails to correctly identify adrenal insufficiency in early/acute and middle/subacute stages of sepsis.

The ACTH test augments inflammatory cytokine production. Unexpectedly, we found that the ACTH test rapidly and significantly increased IL-6 levels in circulation. Under physiological conditions, the ACTH test increased IL-6 levels to 0.2 ng/mL and 0.5 ng/mL at 1.5 and 3 hours after ACTH stimulation, respectively (Figure 2A). Under septic conditions, the ACTH test increased IL-6 to lethal levels, from 15.7 ng/mL to 23.4 ng/mL at 3 hours after CLP treatment (Figure 2B). We also investigated the effect of ACTH at a higher dose (4 IU) on cytokine production during the early stage of sepsis. Three or 24 hours after CLP, ACTH triggered more significant increases in multiple inflammatory cytokines, including IL-6 (Figure 2, C–H).

ACTH test augments inflammatory cytokine production. (A) C57BL/6J mice were treated with 0.1 IU ACTH. Serum IL-6 levels were quantified at the indicated times (n = 6). (B) C57BL/6J mice were challenged with CLP for 3, 24, and 48 hours and treated with 0.1 IU ACTH (n = 6). Serum IL-6 levels were quantified 1 hour later. (C–H) C57BL/6J mice were challenged with CLP for 3 and 24 hours and treated with 4 IU ACTH (n = 7–9). Serum cytokines were quantified 1 hour later by the 31-plex cytokine panel method. *P < 0.05; **P < 0.01; ****P < 0.0001 by 1-way ANOVA with Tukey’s multiple-comparison correction.

Together, we demonstrated that the ACTH test induces a significant increase in inflammatory cytokine production, to lethal levels under septic conditions.

The ACTH test augments inflammatory signaling in the adrenal gland through transcriptional regulation of AP-1. We next investigated the mechanisms underlying ACTH promoting cytokine production. We first searched for the organ that produces IL-6 by quantifying IL-6 mRNA expression in different organs. Unexpectedly, among tested organs, we only observed a significant increase in IL-6 mRNA expression in the adrenal gland upon ACTH stimulation (Figure 3A). We then performed RNA-seq analysis to further investigate how ACTH stimulates inflammatory signaling. Compared with CLP mice, ACTH-treated CLP mice displayed 424 significantly differentially expressed genes, with 317 genes upregulated and 107 genes downregulated (Figure 3B). Among these, a number of inflammatory cytokines were significantly upregulated, including Il6, Lif, Cxcl1, Cxcl2, and Il1b (Figure 3C). KEGG enrichment analysis revealed activation of multiple signaling pathways in the ACTH-treated group (Figure 3D). The activation of complement and coagulation cascades and inflammatory signaling pathways is expected to contribute to worse outcomes in ACTH-treated mice. We evaluated the top 21 differentially expressed genes in the ACTH-treated group compared with the PBS-treated group. We found a group of transcription regulators among the top 21 differential expressed genes (Figure 3E). We then utilized Ingenuity Pathway Analysis (IPA) to assess the upstream transcription regulators. We found the upregulation of several transcription regulators in the ACTH-treated group, including early growth response factor (EGR) (Egr1, Egr2, Egr3, and Egr4), activator protein-1 (AP-1) (Fosb, Fos, Junb, and Atf3), and Krüppel-like factor 4 (Klf4) (Figure 3F). Inflammatory genes, including Il6, have been shown to be regulated by several transcription factors such as NF-κB, AP-1, cAMP response element (CRE)–binding protein (CREB), and CCAAT-enhancer-binding proteins (C/EBPs) (34, 35). We looked at the expression of these transcription regulators and found a significant upregulation of AP-1 family members (Fosb, Junb, and Fos) (Figure 3G), but no significant changes in the expression of NF-κB, CREB, and C/EBPs families (Figure 3, H–J). An early in vitro study showed that ACTH increases IL-6 release from cultured rat adrenal cells (36) and another in vitro study showed that ACTH induces the gene expression of Fos and Junb in cultured bovine adrenal fasciculata cells (37). These studies support our conclusion that ACTH augments cytokine production in the adrenal gland through AP-1 signaling.

ACTH test augments inflammatory response in the adrenal gland through transcriptional regulation of AP-1 in sepsis. C57BL/6J mice were challenged with CLP (25G, full ligation) for 3 hours, and then treated with PBS or 4 IU ACTH. After 45 minutes, RNA from different tissues was isolated. (A) IL-6 mRNA expression was quantified by qRT-PCR. IL-6 relative mRNA expression was normalized to U36B4 expression and analyzed using a 2-tailed Student’s t test. (B–J) Adrenal RNA-seq analysis. (B) Volcano plot. (C) Differentially expressed cytokine genes. (D) The top 6 activated signaling pathways by KEGG enrichment analysis. (E) The top 21 differentially expressed genes in the adrenal gland. (F) The upstream transcriptional regulators analyzed by IPA. (G–J) Graphs showing upregulation of AP-1 signaling but not other signaling pathways (n = 3). *P < 0.05, **P < 0.01. RNA-seq analysis was performed using pairwise comparisons between conditions, applying a negative binomial generalized log-linear model with the glmLRT function in edgeR (https://bioconductor.org/packages/release/bioc/html/edgeR.html). (K) Schematic model of ACTH triggering inflammatory cytokine production in the adrenal gland.

Taken together, our results demonstrated that the ACTH test augments inflammatory cytokine production in the adrenal gland through AP-1 signaling (Figure 3K).

The ACTH test augments inflammatory cytokine production in septic mice with RAI. As an HDL receptor, SR-BI mediates the uptake of cholesterol from HDL, which is required for iGC synthesis under stress conditions (24, 26–32). Therefore, the adrenal gland–specific SR-BI–null (SF1CreSRBIfl/fl) mouse is a unique RAI model. Upon CLP challenge, wild-type (SRBIfl/fl) mice exhibited a normal adrenal stress response, indicated by stress levels of iGCs (Figure 4A) and well-controlled IL-6 (Figure 4B) in circulation. In contrast, SF1CreSRBIfl/fl littermates showed adrenal insufficiency, with no iGC production (Figure 4A) and uncontrolled IL-6 (Figure 4B) in circulation. We then performed the ACTH test on CLP-challenged mice. At the early (CLP 3 hours) and middle stages (CLP 24 hours) of sepsis, almost all SRBIfl/fl mice had a ΔGC of less than 90 ng/mL upon 0.1 IU of ACTH test (Figure 4, C and D). Even at CLP 48 hours, 1 out of 3 SRBIfl/fl mice had a ΔGC of less than 90 ng/mL upon 0.1 IU of ACTH test. Thus, despite the wild-type mice having a normal adrenal stress response to sepsis and a well-controlled inflammatory response, they were incorrectly identified as RAI/CIRCI by the ACTH test at early, middle, and even late stages of sepsis. As expected, the ACTH test did not induce more iGC production in SF1CreSRBIfl/fl under septic conditions (3, 24, and 48 hours after CLP) (Figure 4, C and D). Three hours after CLP, we treated the SF1CreSRBIfl/fl mice with different doses of ACTH (0.1, 0.2, 0.3, and 4 IU). The ACTH treatment at 0.1 IU increased IL-6 to lethal levels, from 20 to 40 ng/mL, and the ACTH treatment at 4 IU increased IL-6 to 60 ng/mL in SF1CreSRBIfl/fl mice (Figure 4E). In addition to IL-6, the 31-plex analysis showed that ACTH significantly increased 18 inflammatory cytokines in SF1CreSRBIfl/fl mice (Figure 4F).

ACTH test augments inflammatory cytokine production in septic mice with RAI. (A and B) SRBIfl/fl and SF1CreSRBIfl/fl mice were challenged with CLP (25G, half ligation), serum corticosterone was measured at 3, 24, and 48 hours after CLP, and IL-6 levels were measured at 3 and 24 hours after CLP (n = 5–10). (C and D) SRBIfl/fl and SF1CreSRBIfl/fl mice were challenged with CLP (25G, half ligation); 3, 24, and 48 hours later, the mice were treated with 0.1 IU ACTH. Corticosterone was measured before and 1 hour after ACTH treatment (n = 5–10). (E) SF1CreSRBIfl/fl mice (n = 3–19) were challenged with CLP for 3 hours and then treated with different doses of ACTH (0.1, 0.2, 0.3, 4 IU). Serum IL-6 levels were quantified 1 hour later. (F) SRBIfl/fl (n = 5) and SF1CreSRBIfl/fl mice (n = 10) were challenged with CLP (25G, half ligation); 3 and 24 hours later, the mice were treated with 4 IU ACTH and serum cytokines were measured 1 hour later with the 31-plex cytokine method. *P < 0.05; **P < 0.01; ***P < 0.001 by 1-way ANOVA with Tukey’s multiple-comparison correction (A–F). In F, *P < 0.05, **P < 0.01 for SF1CreSRBIfl/fl mice; #P < 0.05 for SRBIfl/fl mice.

Taken together, these results demonstrated that ACTH test triggers more inflammatory cytokine production in septic mice with RAI.

The ACTH test moderately decreases survival in septic mice. Excessive production of inflammatory cytokines is associated with poorer outcomes in sepsis (38, 39). In this study, we assessed the impact of the ACTH test on the survival of SRBIfl/fl and SF1CreSRBIfl/fl mice. The ACTH test moderately reduced the survival rate from 89% to 75% in wild-type mice (Figure 5A) and from 48% to 23% in RAI mice (Figure 5B). We conclude that the ACTH test may have detrimental effects, as it significantly increases inflammatory cytokine production and moderately reduces survival in septic mice, regardless of RAI status.

ACTH test moderately decreases survival in septic mice. SRBIfl/fl (A) and SF1CreSRBIfl/fl mice (B) were challenged with CLP for 3 hours (23G, half ligation in SRBIfl/fl mice; 27G, one-third ligation in SF1CreSRBIfl/fl mice). Then, mice were treated with PBS or 0.1 IU ACTH. Survival was monitored for 7 days and analyzed by log-rank test. The terms half ligation and one-third ligation refer to the points of ligation on the cecum based on the distance from its distal end.