Broiler chickens grown under intensive breeding conditions commonly are infected by diverse pathogenic and nonpathogenic microorganisms. These infections frequently induce immune stress in broilers. Immune stress may cause abnormal changes in the behavior and metabolism of broilers which lead to the decline of immunity and disease resistance and ultimately result in growth inhibition. Thus, immune stress is a key factor that hinders broilers from achieving optimal rates of feed and ideal growth and thereby causes significant damage to the poultry breeding industry (1–3). Immune-stressed broilers produce increased levels of inflammatory cytokines which induce fever and anorexia (4, 5). Moreover, these inflammatory cytokines activate the hypothalamic-pituitary-adrenal (HPA) axis and stimulate the adrenal gland to secrete corticosterone (CORT) which accelerates the oxidative decomposition of muscle (6, 7). Despite these intriguing observations, the mechanism by which growth is restrained in immune-stressed broilers has not been elucidated fully.

Expression of cyclooxygenase-2 (COX-2), a key enzyme in the biosynthesis of prostaglandins (PGs), is enhanced in the hypothalamus following stimulation by lipopolysaccharide (LPS) (8, 9). LPS is an intrinsic component of the outer membrane of Gram-negative bacteria and is a potent activator of the immune system. Nevertheless, LPS-induced decrease in feed intake of stressed broilers was ameliorated significantly by treatment with the COX-2 inhibitors celecoxib or indomethacin (10), which suggests that immune stress-induced anorexia may be mediated by COX-2. PGs are inflammatory mediators that are elevated in the brain during inflammation and which modify key aspects of neuronal activity to evoke symptoms of sickness behavior (11). Prostaglandin H₂, the end product of COX enzyme activity, is converted into diverse PGs, including PGD₂, PGF_2α, PGE₂, PGI₂, and thromboxane A2 (12), each of which exerts specific physiological effects. Interestingly, PGE₂ was the most potent PG that induced anorexia by suppressing food intake in mice via the EP4 receptor. This receptor is one of four receptor subtypes for PGE₂ (13, 14). However, the mechanism by which PGE₂ regulates food intake in broilers remains to be explored. We previously demonstrated that the expression of COX-2 and the downstream mPGEs-1 gene that encodes microsomal prostaglandin E synthase-1, which is a terminal PGE2 synthase, promoted the excitability of hypothalamic corticotropin-releasing hormone nerve cells and activation of the HPA axis (15, 16). These data led us to test the potential involvement of COX-2 and its major downstream product PGE₂ in growth inhibition of broilers under immune stress conditions. Therefore, in this study we constructed a broiler immune stress model by intraperitoneal injection of LPS to investigate stress-induced expression of the COX-2 signaling pathway and the cascade of PGE₂ receptors (EP receptors) in the hypothalamus.

LPS-induced immunological stress activates persistent pro-inflammatory responses that contribute to degradation of production performance in broilers (24–26). However, the molecular mechanisms that underpin growth inhibition caused by immunological stress remain elusive. The purpose of this study was to investigate the mechanism by which immunological stress restrains broiler productivity. We found that the performance of broilers that were stressed by LPS challenge was reduced which was reflected in a decrease of body weight gain 24 h after the first LPS injection as well as a reduction in feed intake and in the jejunum index, particularly 72 h after LPS administration. The HPA axis is activated when broilers are exposed to stressors which results in a sharp rise in CORT concentrations (27). Thus, CORT levels provide a reliable indicator of chicken stress status. In agreement, we found that LPS challenge increased CORT concentrations in the serum of broilers which indicates that the broiler immune stress model was valid. In addition, we observed that LPS-induced stress elevated TNF-α levels in serum and decreased the thymus index. TNF-α is a key cell signaling protein in the inflammatory response and induces apoptosis in diverse cell types (28). The observations here indicate that immunological stress may stimulate an inflammatory response and inhibit thymus development which subsequently leads to a decline in broiler immune function. Based on the preceding results, we speculate that the primary cause of LPS-mediated growth inhibition of broilers involves enhancement of the synthesis and secretion of stress hormones. These hormones may stimulate signal molecule receptors that are implicated in appetite control and growth which promote increased catabolism, anorexia, and growth inhibition. Alternatively, pro-inflammatory cytokines that are produced as a consequence of immunological stress may repartition nutrients and increase the catabolic activities that are associated with broiler growth.

The hypothalamus plays a key role in the regulation of feeding (29). The hypothalamic nuclei involved in feeding control include the arcuate nucleus (ARC), paraventricular nucleus, ventromedial nucleus, dorsomedial nucleus, and lateral hypothalamic area. These loci form a regulatory network for feeding activities and maintenance of energy metabolism (30, 31). ARC is the most critical nucleus for feeding regulation (32). Two principal neuronal populations that modulate appetite conversely exist in the ARC: AgRP neurons which co-express NPY, and POMC neurons. Activation of POMC decreases daily feed intake, whereas stimulation of AgRP/NPY boosts feed intake (33–36). Here we found that the feed intake of broilers was reduced 72 h after the first LPS challenge and that the expression of POMC in the hypothalamus of these animals was increased. The expression of NPY also was elevated compared with the control group which is inconsistent with previous studies (37–40). We speculate that expression of the SOCS3 gene was enhanced in the hypothalamus of stressed broilers within 2 h of the primary LPS injection. The SOCS3 protein is the main inhibitor of leptin and insulin signaling which causes cellular leptin and insulin resistance and dampens energy production (41–43). This state of energy deficiency may increase NPY expression temporarily in the hypothalamus. Thus, our results indicate that POMC may be the key regulatory factor in LPS-induced anorexia which was reflected in the decreased feed intake of broilers 72 h after LPS administration. However, the effect of LPS on NPY expression requires further exploration.

The hypothalamic-pituitary-growth hormone axis plays a crucial role in the regulation of growth (44). The hypothalamus secretes GHRH and SST hormones which act on the pituitary gland with opposing effects to modulate growth hormone (GH) secretion: GHRH promotes GH secretion whereas SST inhibits GH secretion. GH enters the blood and binds to the liver growth hormone receptor to stimulate the synthesis and secretion of IGF-1 by liver cells. IGF-1 interacts with binding proteins on target cells and tissues and enhances the growth and development of skeletal muscle and the cardiovascular and cerebrovascular systems (45, 46). GHRH may be attenuated by a pronounced increase in SST concentrations or, conversely, SST may be potentiated by GHRH stimulation (47). Although stimulation of GHRH appeared to predominate compared with SST stimulation in response to LPS in this study, we found that body weight gain of broilers in the immune stress group was reduced at 24 h after the first LPS injection. Moreover, the decline of GHRH secretion in the hypothalamus was accompanied by a decrease of IGF-1 levels in serum 2 h after LPS challenge. However, the expression of SST in the hypothalamus of stressed broilers did not fluctuate. Therefore, the growth inhibition of stressed broilers partly may reflect inhibition of the activity of hypothalamic GHRH neurons which negatively regulate the hypothalamic-pituitary-growth hormone axis which in turn leads to the decrease of production performance. Thus, immune stress decreased GHRH secretion and increased POMC secretion which reduced feed intake and slowed the body weight gain of stressed broilers. However, the mechanism by which immune stress affects GHRH and POMC expression in stressed broilers needs further investigation.

The NF-κB and p38/MAPK signaling pathways drive the expression of inflammatory mediators and enzymes involved in the inflammatory response, including COX-2 (48, 49). COX-2 is a key enzyme in the epoxidase pathway of the arachidonic acid metabolic network and expression of COX-2 profoundly affects the production of PGs (12). Here, we observed that LPS administration activated the MAPK-NF-κB pathway though the TLR1 receptor and MyD88, and increased the secretion of COX-2 and mPGEs-1 in the hypothalamus of stressed animals. The enhancement of COX-2 expression preceded mPGEs-1 expression. Recent studies have revealed that COX-2 participates in the production of LPS-induced proinflammatory factors by activating microglia-related signaling pathways (50). We found here that COX-2 was released early and produced at the same time point as IL-1β and CD40 in the hypothalamus of immune-stressed broilers. Thus, COX-2 may participate in the production of IL-1β by activating microglia-related signaling pathways. The IL-1β cytokine induces PGE₂ production via up-regulation of COX-2 and mPGEs-1 expression in several cell types (51). In addition, the function of PGE₂ mainly is mediated by G protein-coupled receptors located in the cell membrane. EP receptors comprise four subtypes (EP1, EP2, EP3, and EP4) (52). Here, the EP4 gene was significantly up-regulated in stressed broilers whereas expression of EP1, EP2, and EP3 did not change significantly. Genome-wide association studies previously revealed that polymorphisms in the EP4 gene are associated with overexpression of EP4 and a more severe disease phenotype in patients with irritable bowel disease (53–55). Moreover, certain variants in the EP4 gene are associated with Crohn’s disease (56). These observations suggest that COX-2-PGE₂-EP4 signaling also may participate in the pathogenesis of inflammation. These findings suggest that IL-1β induced by LPS regulates the key components of the PGE₂ biosynthetic cascade, e.g., COX-2, mPGEs-1, and EP4, through the MAPK-NF-κB pathway. The combination of PGE₂ and EP4 receptor in microglia may trigger a positive feedback loop that promotes the amplification of COX-2 effects during LPS challenge. Therefore, the decrease in growth performance of broilers under immune stress is associated strongly with COX-2 signaling mediated by the NF-κB/MAPK signaling pathway.

The immune and neuroendocrine systems are connected closely. Inflammatory cytokines induced by LPS directly contact microglia in the hypothalamus and promote production of increased concentrations of COX-2 by these cells. The level of PGE₂ increases as a consequence and this PG diffuses to the feeding center to cause animal anorexia and inhibition of food intake (57–59). Analogously, other studies found that the application of COX-2 inhibitors alleviated the loss of appetite caused by LPS in poultry (10) which suggests that COX-2 may mediate the suppression of feed intake of animals under immune stress. Deletion of the gene for EP4 in microglial cells reduced microglial-POMC neuron contacts (60) and implicated PGE₂ signaling via EP4 as an important regulator of microglia-POMC neuron interactions. In addition, accumulating evidence has substantiated that IL-1 affected hypothalamic peptide release were mediated via COX stimulation of PGE synthesis. IL-1-induced stimulation of GHRH and SST are antagonized by indomethacin which inhibits PG synthesis via the COX pathway (61). In view of these observations, we speculate that LPS-induced growth inhibition may be mediated by the COX-2-mPGEs-1-PGE₂ axis in the hypothalamus: PGE₂ binding to EP4 receptors via appetite and growth regulatory peptides thereby may affect growth and development in stressed broilers.

The molecular mechanism of stress-activated COX-2 signaling in growth regulation of broiler chickens was probed further by establishing a celecoxib pretreatment group. Celecoxib is a new, non-steroidal anti-inflammatory drug that selectively inhibits COX-2 enzymatic activity and the subsequent pro-inflammatory factor, PGE₂ (62). Clinical studies demonstrated that celecoxib passes through the blood-brain barrier (63). Our results here indicate that pretreatment with celecoxib increased the body weight gain of stressed broilers, and also resolved inflammation in the serum and hypothalamus. Moreover, CD40 levels also were reduced which suggests that microglia displayed a less activated phenotype (64). In addition, celecoxib significantly down-regulated the expression of CX3CL1, CCL4, IRF7, LY96, MAP3K8 and TLR1B in the MAPK-NF-κB signaling pathway. These observations verify our previous results in this study which indicated that COX-2 may act as an important mediator in the activation of the AMPK-NF-κB signaling pathway via the EP4 receptor. Furthermore, our finding that inhibition of COX-2 activity elevated GHRH expression in the hypothalamus of stressed animals without affecting SST expression is consistent with our previous inference that COX-2 signaling plays an important role in LPS-induced growth inhibition by binding to EP4 receptors on GHRH neurons to moderate GHRH expression. It is important to note that we observed that inhibition of COX-2 activity increased NPY expression but exerted no detectable effect on POMC. Combined with previous findings, these data imply that LPS-induced anorexia may derive from the concerted action of NPY and POMC. COX-2 signaling may inhibit growth in stressed broilers by binding to EP4 receptors on NPY neurons, in which case the activity of POMC neurons may not be impacted by the COX-2 pathway. LPS causes inflammation systematically and the NF-кB signaling pathway interacts directly with POMC to promote the transcription and expression of the POMC gene (65). Therefore, the decline in food intake among stressed broilers caused by the up-regulated expression of POMC may be mediated by the NF-кB signal pathway in our study.

In conclusion, this study suggests that immune stress regulates the MAPK-NF-κB signaling pathway and drives the transcription of inflammatory factors such as COX-2 ( Figure 10 ). COX-2-PGE2 signaling induces the secretion of related inflammatory factors via the EP4 receptor on microglia. Moreover, PGE2-EP4 also may cascade with the appetite regulatory peptide NPY and the growth regulatory factor GHRH to dampen the concentration of IGF-1 in the serum of stressed broilers. We propose that this relay in turn decreases feed intake and body weight gain and leads to the suppression of growth of immune stressed broilers.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number (s) can be found below: PRJNA940969 (SRA).

The animal study was reviewed and approved by Animal Care and Use Committee of Henan University of Science and Technology.

Investigation, KL, HT, and XH. Methodology, KL, XH, and HT. Software, KL, HT, and WZ. Formal analysis, KL, YZ, DB, and WZ. Data curation, KL, HT, YQL, and YHL. Writing the original draft, KL, WZ, and DB. Reviewing and editing, DB, KI, WZ and BZ. Supervision, YM and project administration, YM. All authors contributed to the article and approved the submitted version.