Poultry production and performance is affected by various factors including exposure to biotic and abiotic stressors. Most of these stressors, affects the immune homeostasis of the poultry birds leading to poor production performance and mortality. Hence, its important to modulate the host immune response, to obtain optimal performance. Phytogenics is one of the effective and important immune modulators, used to overcome immune stress. These are capable of acting in host, directly and/or indirectly to modulate its immune response and maintain immune balance, on exposure to various stressors. These phytogenics, act by different modes including, interaction with different membrane proteins, signalling pathways, transcription factors, genes and mediators. This review discusses such different mechanisms of host directed immunomodulatory activity of phytogenics in poultry.
Immune stress is one of the major factors influencing the growth performance, infection control, mortality, organ growth etc, in poultry production. Immune stress is loss of immune homeostasis caused by various factors including different production and environmental stressors. For instance, infections and exposure to environmental stressors such as low or high temperature and seasonal variations etc, leads to loss of innate and acquired immune responses. Similarly, exposure to toxins such as fusarium also supress immune function and increases the susceptibility to pathogens. These stressors supress the immune response by reducing the immune cell production and proliferation, development of immune organs such as spleen, bursa and thymus, affect cellular trafficking, proliferation, cytokine secretion, antibody production, phagocytic and cytolytic activity. Also, as a result of these stressors, there is marked increase in ROS production, oxidative damage and increase in inflammatory reaction such as, infiltration of inflammatory cells and activation of inflammatory cascade. All these results in loss in energy, decrease in production performance and may also lead to mortality (Lara and Rostagno, 2013; Rosales, 1994; Yang et al., 2011). Hence, its important to modulate immune responses of birds and maintain immune homeostasis, to achieve optimal growth performance in poultry.
Phytogenics are natural plant-based substances used effectively in animal health and nutrition to improve bird’s health, growth and performance. Many of such plant extracts, essential oils and phytochemical compounds were proven to have immunomodulatory activity in poultry birds. Phytogenics, have shown to be effective in modulating innate/ non-specific and humoral/specific immune responses. Some of such phytogenic agents or extracts, which is reported to have immunomodulatory activity include, Curcuma longa, Capsicum annuum, Cinnamomum verum, Allium sativum, Withania somnifera, Tinospora cordifolia, Echinacea sp. Salvia officinalis, anise (Pimpinella anisum), oregano (Origanum vulgare), caraway (Carum carvi), thyme (Thymus vulgaris), rosemary (Rosmarinum officinalis), clove (Syzygium aromaticum), camomile (Chamomilla recutita), and fennel (Foeniculum vulgare) (Catanzaro et al., 2018; Karaskova et al., 2015; Singh et al, 2016; Swaggerty et al., 2019; Singh et al, 2016).
Immunomodulatory activity of phytogenics depends on various factors including, nature of stressors, dosage, type of extracts and formulation used, phytochemical constituents and so on. Mechanism of action also varies from one phytochemical to another. In general, phytochemicals are shown to have multi-level immune-modulatory activity starting from its interaction with intestinal receptors, signalling proteins, transcription factors and inflammatory mediators. They also stimulate development and functioning of immune organs such as spleen, bursa and thymus. In most instance, multiple site of action of these phytochemicals results in better immunomodulatory activity. General immunomodulatory mechanism of certain phytochemicals proven to be effective in poultry birds, has been discussed in this review.
Modulating Expression of Heat Shock Proteins (HSPs):
HSPs are membrane proteins expressed more in various stress conditions including, thermal, pH, hypoxia, pathogen encounter etc. During acute heat stress, normal cellular functions are compromised and HSPs activates cellular responses to protect the cells from oxidative damage, by increasing the expression of anti-oxidant enzymes. HSPs also facilitate immune response by playing an important role in antigen presentation, cross-presentation (followedby inducing cytotoxicity T-cell responses and thereby eliciting pro-inflammatory cytokine production), activation of macrophages & lymphocytes and phagocytes especially macrophages and dendritic cells. All these leads to energy loss, cellular damage due to inflammatory reaction and ultimately affects birds performance (Tsan and Geo, 2009). Extracts of Curcuma longa, Allium sativum, Zingiber officinale and few others have found to modulate immune response via modulation of heat shock proteins (Hashemi and Davoodi, 2012). For instance, in a study conducted by Zang et al (2015), C. longa extract supplementation decreased the expression of HSP 70 in breast muscle of broilers. HSP70 protein plays a major role during thermal and oxidative stress. This stress response protein also mediates cascade of immune reactions in the cell to prepare itself to defend against the stress and helps maintain thermal homeostasis. C. longa supplementation not only help overcome oxidative stress by acting as an anti-oxidant, it also modulates the HSP mediated cellular stress response by altering the mRNA levels of HSP, there by alters the downstream immune response.
The change in HSP levels and anti-oxidant enzymes depends on various factors including the type of tissue, age of birds, ROS concentration, stress exposure time and dose of phytogenics. Table 1 summarizes few HSP mediated immunomodulatory activity of phytogenics in poultry birds.
Modulating Expression of Toll Like Receptor (TLRs):
TLRs are one of the major innate immune pattern recognition receptors, which play major role in infection defence and immune homeostasis. Atleast 10 different TLRs have been identified so far in chickens TLR1A, 1B, 2A, 2B, 3, 4, 5, 7, 15 and 21. TLRs has been identified in macrophages, heterophils and B cells, where they mediate host-responses to pathogens by promoting cellular activation and the production of cytokines. In response to pathogen invasion and infection, TLRs elicits ROS production, inflammatory cytokines and upregulate inflammatory reaction and also elicits host adaptive immune response (Hang., 2017; Keestra et al., 2013; Kannaki et al., 2010). It is important to modulate the expression of TLR and/or LPS oligomerisation of TLRs, to prevent excess energy loss and host cell damage. Several phytogenics have shown to modulate the expression of TLRs and/or prevent oligomerization of TLRs by pathogen-lipopolysaccharides. Down-regulation results in attenuation of host inflammatory reactions. For instance, Du et al. (2016), reported increase in mRNA expression of TLR-2 protein, during C. perfringens infections in broilers. Supplementation of thymol and carvacrol resulted in marked decrease in the mRNA expression of TLR-2 protein, in addition to considerable improvement in pathogen control, lesion score and bird’s performance. Also increase in NDV anti-body titre was observed, which is indicative of improvement in humoral immune response. Similarly, Nawab et al (2019) also reported significant decrease in TLR-4 (an essential component of innate and adaptive immune signalling) mRNA expression, upon curcumin supplementation in layers during heat stress. Marked TLR-4 down regulation resulted, decrease in inflammatory protein gene expression and decrease in cytokines such as IL-6, IL-1β and TNF-α, thereby reducing the inflammatory reactions and tissue damage. Thus, curcumin was proven to improve immunity and heat stress by modulating TLR-4 expression.
Summary of selected phytogenics role in TLR expression of poultry birds, up on oral administration, has been briefed in Table 1.
Table 1: Immunomodulatory phytochemicals and its effect on HSPs and TLRs of poultry birds.
Effect on Immune cells – Activation & Proliferation:
Immune cell proliferation, activation and signalling between them forms the basis of host immune response including innate and adaptive immune responses. Phytochemicals modulates immune cell function including cells such as neutrophils, macrophages, monocytes, natural killer cells (NK cells), dendritic cells (DCs), T cells, and B cells. Phytogenics inhibits PHA-induced T-cell proliferation, interleukin-2 (IL-2) production, nitric oxide (NO) generation, and lipopolysachharide-induced nuclear factor-kappaB (NF-kappaB) mediated inflammatory pathway and augments NK cell cytotoxicity. It also, inhibits NFkB and MAPK signalling resulting in inhibition of stimulatory signal necessary for T cell activation. It also impairs production of pro-inflammatory cytokines (such as IL-12). In several, in vitro and in vivo studies, curcumin confirmed to have above activities (Catanzaro et al., 2018; Yadav et al., 2015). Many plant extracts including, Curcuma longa, Capsicum annuum, Cinnamomum verum, Allium sativum, Withania somnifera, Tinospora cordifolia, Echinacea sp. Salvia officinalis, anise (Pimpinella anisum), oregano (Origanum vulgare), caraway (Carum carvi) and thyme (Thymus vulgaris) were reported to modulate immune response of host by modulating immune cell activation and proliferation (Catanzaro et al., 2018; Karaskova et al., 2015; Singh et al, 2016; Swaggerty et al., 2019; Singh et al, 2016).
Phagocytosis is a critical part of innate defence mechanism. Many different immune cells including neutrophils, monocytes, macrophages, mast cells, and dendritic cells, perform phagocytosis. Purpose of phagocytosis is not just evasion of incoming pathogens but also to communicate to lymphocytes about the incoming pathogen by antigen presentation via MHC glycoproteins. This process also elicits inflammatory reactions thorough secretion of inflammatory mediators. Thus, modulating activity of phagocytic cells is one of the important strategies to alter the immune response of the host. Supplementation of Salvia officinalis essential oil in broiler chickens resulted in decrease in oxidative stress and phagocytic activity. Supplementation of Thymus vulgaris, rosemary (Rosmarinus officinalis) and fenugreek (Trigonella foenum graecum) extract also resulted in similar decrease in phagocytic activity, additionally improvement in white blood cells and other haematological parameters (Karaskova et al., 2015). In vitro studies conducted using Echinecea sp. resulted in increase in the activation of macrophages, expression of MHC II molecules and chemokines such as IL-1_, IL-6, IL-12p70, TNF-α and NO. All these, resulted in enhanced phagocytic activity (Catanzaro et al., 2018). Thus, phytogenics can be used to both enhance and suppress the activity of phagocytic cells, based on the requirement.
Modulation of inflammatory mediators:
Interleukins, cytokines, tumor necrosis factor-α are major inflammatory mediators secreted by immune cells, to orchestrate the inflammatory reaction. NO and TNF produced by macrophages plays a major role in inflammatory process by acting as a mediators to interact with other immune cells. TNF mediates the T cell activation, granuloma formation during pathogen invasion. Transcription factor NFkB, also facilitate the expression of inflammatory genes, during stress and infection. In addition to these, signalling pathway modulated by MAP kinases and STAT3 transcription factor also plays a major role in production and release of pro-inflammatory cytokines. It’s well known that inhibition of signalling pathways and/or mediators, leads to reduction in downstream inflammatory reactions. Curcumin proven to reduce lipopolysaccharide (LPS)-induced expression of IL-6, TNF-α and prostaglandin-endoperoxide synthase 2 by modulating STAT3 and MAPK signalling pathway. Modulation of NFkB by phytochemicals such as Curcumin results in reduction of expression of inflammatory mediators such as COX-2, inducible nitric oxide synthase (iNOS) and interleukins (Catanzaro et al., 2018; Yadav et al., 2015). Carvacrol inhibits transcription factor NFkB and MAPK signalling leading to decrease in IL-1β, IL-6, IFN-γ and TNF-α, there by modulate the inflammatory reactions (Huang and Lee. 2018). Lee et al (2011), reported increase in lymphocyte proliferation and increase in IL-1b, IL-6, IL-15 and interferon-γ upon cinnamaldehyde supplementation, in coccidiosis infected birds. These in turn influenced the immune response of birds against coccidiosis infection. Carvacrol, capsaicin and cinnamaldehyde supplementation resulted in significant decrease in inflammatory cytokine production and changes in immune related gene expression. These phytochemicals supplementation also, resulted in suppression of nitric oxide (NO) production, up-regulation of co-stimulatory molecules (CD80 and CD69) and proinflammatory cytokines such as TNF-α and IL-1β. All these indicates the role of these phytochemicals in control of inflammation and immune modulation (Kim et al., 2010).
All these results infer us that supplementation of phytogenics modulates the activity and expression of immune modulators to control inflammatory reactions in infected and/or birds under going stress.
Modulation of Anti-Body Production:
Upon encounter of pathogens or antigens, T cells and B cells mediate the synthesis and secretion of specific antibodies, which forms the basis of humoral and adaptive immune response. Several inflammatory mediators including lymphokines, cytokines, interleukins and interferons mediates this process. Saponins, phytosterols, flavonoids and terpenes of plant such as Ginseng and Ginko biloba stimulates the production of chemokines such as IL-2, IL-6, TNF-α and INF-γ, and also stimulates macrophage activation and lymphotcyte activity which in turn affects the humoral immune response (Hashemi and Davoodi. 2012). Zaki et al (2016) reported elevated levels of anti-bodies titre levels against new castle disease virus (NCD) upon supplementation of coriander seed, turmeric and thyme supplementation. Curcumin supplementation in broiler birds, significantly increased the serum immunoglobulins IgA and IgM production (Emadi and Kermanshahi, 2007). The increase in anti-body production upon supplementation of phytogenics, probably may be through modulation of expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ) thereby enhancing the B cell proliferation, plasma cell differentiation and antibody production (Oshima et al, 2012; Xu et al., 2018). Echinacea purpurea, oregano, thyme, cinnamon and turmeric were confirmed to increase the antibody titre and lymphocyte count. Oregano supplementation increased the anti-body titre of NDA and influenza vaccinated broiler birds. Similarly, Echinaea sp. extract supplementation, in broiler birds, increases the anti-body titres to IBD vaccine, in addition to increase in IL-2 and TNF-α production (Catanzaro et al., 2018; Huang and Lee. 2018; Ma et al., 2009).
Modulation of Intestinal Morphology and Gut microbiota:
Its well known that gut and its associated microflora plays a major role in the development and functioning of healthy immune response, particularly innate or non-specific immune response. The first line of immune defence against pathogenic organisms, begins in the intestine. Gut and GALTs (Gut Associated Lymphoid Tissues) acts as a first line defence by forming an interface between the intestine, neuro-endocrine system and host immunity. Both innate and acquired host immune response begins in the intestine, while encountering pathogens. Mucosal lining, underlying epithelium comprising enterocytes, mucus producing globlet cells and endocrine cells forms the basis of first line immune defence mechanism. Tight junction intestinal epithelial lining, mucus secretion, secretory IgA and antimicrobial peptides aids control of invading pathogens. In addition to these, microbiota of the host is one of the crucial factors affecting the host immune response. It’s important to maintain beneficial gut microflora in order to help healthy interaction of intestinal microflora and host immune response, control of pathogen growth, promote digestion and nutrient absorption and maintain gut integrity. Dysbiosis have shown to increase pathogen load, susceptibility of host to infection, immune suppression, poor gut integrity etc. Hence, its very essential to maintain gut integrity and beneficial gut microflora to sustain healthy immune response and performance of the host (Garcia-Garcia et al., 2013; Kamada et al., 2013; Kim and Lillehoj, 2019).
Phytogenic feed additives were reported to positively influence the intestinal morphology such as increasing in villi length, gut integrity and modulate gut microflora, which in turn improves the immune response and performance of the host. Reports suggested that curcumin supplementation significantly improves, intestinal morphological parameters such as intestinal villi length and crypt depth to villi length ratio and also improved the Lactobacillus sp. load in the intestine. These changes might probably the reason for improvement in bodyweight and FCR (Dalal et al., 2018). In another study (Kim et al., 2015), supplementation of capsicum and turmeric oleoresins increased the percentage of Lactobacillus and decreased the pathogen load in intestine, thereby found to be effective in controlling enteritis in commercial broiler birds.
Poultry performance is administered by various biotic and abiotic factors. Immune stress, is one such important factors that should be attenuated to achieve optimal performance in birds. Supplementation of phytogenics is an effective way to overcome immune stress and maintain immune homeostasis, in poultry birds. Several recent studies infer us that, phytogenics have multi-level immunomodulatory activity. Immune modulation starts with the interaction of these compounds with intestinal receptors, followed by modulation of signalling pathways, alteration in gene and protein expression, leading to increase in host immune response, decrease in anti-inflammatory response and resultant change in intestinal morphology and gut microbiota. All these cumulatively aids modulation of host immune response and recovery from immune stress. However, the immunomodulatory activity of phytogenics, shall vary due to various reasons including, dose, formulation, mechanism of action, disease condition, type of stressor etc. Hence, its important to identify and utilize these phytogenics considering all these, to obtain maximum benefits.