The avian immune system can broadly be classified into the innate and adaptive immune systems. The components of the innate immune system are monocytes, macrophages, dendritic cells, neutrophils, and natural killer cells and molecules such as antimicrobial peptides, nitric oxide, cytokines, and chemokines secreted by these cells. The adaptive immune system comprises B-lymphocytes and T-lymphocytes ^[1][105]. The avian lymphoid organs include central immune organs, namely the thymus, bursa of Fabricius, and bone marrow, and the peripheral immune organs are the spleen and cecal tonsils. In addition, there are single or multiple lymphoid follicles dispersed in the lamina propria of the intestine and bronchi, and these are termed gut-associated lymphoid tissue (GALT) and bronchial-associated lymphoid tissue (BALT), respectively ^[2][106].

The gut mucosa and GALT are constantly exposed to antigens of food, gut microbial, and pathogenic bacterial origin. Hence, the principal function of GALT is to prevent the entry and dissemination of pathogens. Non-specific barriers such as peristalsis, lysozyme, antimicrobial peptides, bile salts, gastric secretions, pancreatic secretions, and commensal gut microbiota are the primary defense mechanisms that prevent the entry of pathogens into the gut and pathogen’s systemic dissemination ^[3][107]. The GALT is composed of organized lymphoid tissues consisting of Meckel’s diverticulum, cecal tonsils, payers patches, bursa of Fabricius, regional lymphatics, and mesenteric lymph nodes, and dispersed lymphoid aggregates across the gut lamina propria and intraepithelial leukocytes ^[4][108]. The components of GALT can broadly be classified into mucosal inductive sites that encounter the environmental antigens and mucosal effector sites such as the lamina propria ^[5][109]. The outermost layer of GALT consists of the mucus layer, enterocytes, and intraepithelial lymphocytes separated by a basement membrane from the underlying lamina propria. The lamina propria comprises the submucosa and lymphocytes such as mast cells, dendritic cells, macrophages, natural killer cells, fibroblasts, and the draining lymphatics and lymph nodes ^[5][6][7][109,110,111]. The Payer’s patches are a cluster of B cells creating a follicle divided by T cell-rich segments and randomly dispersed in the wall of the small intestine. The intestinal epithelium overlying Payer’s patches, called the follicle-associated epithelium, consists of specialized cells named microfold cells (M cells), which actively endocytose and pinocytose the gut luminal antigens. The Payer’s patches form a ‘dome’-like structure, and the epithelium is relatively devoid of mucus, enabling effective sampling of the gut luminal antigens and their delivery to the underlying lymphoid tissue ^[8][9][112,113]. The intestinal epithelial cells, intraepithelial lymphocytes, and lamina propria lymphocytes secrete and respond to various cytokines, including TGF-α, TGF-β, IFN-γ, IL-1, IL-2 ^[10][114], IL-3, IL-4, IL-5, IL-6 ^[11][115], IL-8, IL-15 ^[12][116], and GM-CSF ^[10][114].

The gut’s mucosal immune system plays a significant role in the innate immune response to necrotic enteritis. Pathogen recognition receptors (PPRs) present on intestinal epithelial cells and cells of the innate immune system recognize conserved molecular structures on the pathogen, known as pathogen-associated molecular patterns (PAMPs) ^[13][14][117,118]. Toll-like receptors are one of most prominent PRRs expressed on cell surfaces, endocytic vesicular membranes, and other intracellular organelles. PRRs recognize the molecular motifs associated with the pathogen, such as flagellin, lipopolysaccharides, peptidoglycans, and bacterial genetic materials, and initiate innate immune responses to the pathogen. The chicken TLR repertoire comprise 10 genes, of which three are unique to chickens ^[15][119]. The expression of TLRs varies depending on the tissue and cell type, as summarized in Table 14, and this influences the type of immune response elicited in different tissues ^[16][120]. Other important PRRs are the C-type lectin-like molecules, cytosolic receptors, and RIG-I-like receptors ^[17][121].

At the gut mucosal interface, the enterocytes, intraepithelial lymphocytes, and innate immune cells express PRRs which are activated on interaction with the PAMPs, initiating a series of downstream signaling pathways which induce the production of reactive oxygen species, reactive nitrogen intermediates, and proinflammatory cytokines. Activation of the antigen-presenting cells upregulates the expression of co-stimulatory molecules, which are essential for developing adaptive immune responses ^[23][24][127,128]. TLR activation results in the recruitment of adaptor proteins to the intracellular domain of the TLR, further recruiting the interleukin-1 receptor-associated kinase (IRAK). This activates the tumor necrosis factor receptor-associated factor 6 (TRAF6), an essential ubiquitin E3 ligase. TRAF6 subsequently activates transforming growth factor-β-activating kinase (TAK1). TAK1 induces the phosphorylation of IκB or activates the kinases MKK3 and MKK6 and leads to the activation of the transcription factors NFκB and Janus kinase (JNK), respectively. The transcription factors NFκB and JAK translocate to the nucleus and induce the expression of genes necessary for the immune response ^[25][129]. The activation of TLRs on the APCs results in the upregulation of co-stimulators, MHCs and gene expression, inducing pro-inflammatory cytokines and chemokines. These activated APCs migrate to the regional lymphoid organs and facilitate the activation of naïve T-cells ^[26][130]. TLR activation also induces the production of antimicrobial peptides such as β-defensins and cathelicidins, which are the effector molecules of the innate immune system ^[27][131].

In chickens, the cell wall peptidoglycans of Gram-negative bacteria are recognized by TLR2b, TLR4 recognizes the lipopolysaccharide component of Gram-positive bacterial cell membrane, and TLR5 recognizes flagellin of both Gram-positive and Gram-negative bacteria ^[28][132]. C. perfringens challenge increases the expression of TLR1.2, TLR2.1, TLR4, TLR7 and TLR 15 in the spleen and ileum ^[29][133]. Likewise, NE upregulates the TLR2 and TLR15 mRNA expression in the duodenum. In vitro, C. perfringens peptidoglycan significantly increases the mRNA expression of IL-6, IL-8, TNF-α, and iNOS ^[30][134]. These results suggest the importance of TLRs in the innate immune response to C. perfringens challenge.

Activation of the APCs such as dendritic cells, macrophages, and B cells by the binding of ligands to PRRs upregulates the expression of MHC class II molecules and co-stimulators on the APCs. The activated APCs subsequently activate the naive T cells and promote their differentiation to different subsets of T-helper cells ^[31][135]. Activated macrophages secrete IL-12, stimulating the production of IFN-γ, which subsequently induces the differentiation of activated T cells to the Th1 subset, initiating the cell-mediated immune response ^[32][136]. IL-2 secreted by the activated T-cells stimulates effector T cells and regulatory T cells ^[33][137]. A study reported increased mRNA expression of IL-4, IL-10 and IFN-γ in birds challenged with C. perfringens ^[34][60]. The cytokine IFN-γ is associated with macrophage activation, differentiation of naïve helper T-cells to Th1 subset, and expression of MHC class II ^[35][138], whereas IL-4 and IL-10 are immunomodulatory cytokines that suppress the production of proinflammatory cytokines such as IL-12 and IFN-γ ^[36][139].

Interleukin 17A, produced by the Th17 cells, plays a significant role in antimicrobial defense at mucosal barriers by stimulating neutrophil recruitment, the secretion of antimicrobial peptides, and IgA production ^[37][38][140,141]. To compensate for the tissue injury due to infection and inflammation, the cytokines IL-17 and IL-22 promote the regeneration of intestinal epithelial cells. Subclinical NE activates Th2- and Th17-mediated immune responses, characterized by the upregulation of IL-1β, IL-13, and IL-17 cytokine expression in the cecal tonsils and jejunum.

On the other hand, some data suggest that the NE infection is resolved by a Th1 response mediated by the cytotoxic T-cells. The T-cell response to NE infection peaks on day 16, characterized by an increase in the numbers of T-helper cells, cytotoxic T cells, and double positive T cells ^[39][142]. In a study conducted by Park et al. ^[40][143], it was demonstrated that infection with C. perfringens induced the expression of IL-1β, IL-2, IL-12, IL-13, IL-17, IFN-α, IFN-γ, and TGF-β. However, it is noteworthy that the coinfection of Eimeria maxima and C. perfringens reduced the expression of the above-mentioned immune mediators significantly. Expression of iNOS was also significantly reduced in coinfected birds. This can be explained by the fact that the expression of IFN-γ, which mediates the expression of iNOS in epithelial cells and macrophages ^[41][144], is suppressed under coinfection with Eimeria and C. perfringens ^[40][143].

In general, the host immune responses and parameters vary widely between studies on necrotic enteritis models. The variation may be attributed to the different strains of C. perfringens used, modifications of the necrotic enteritis models, and the predisposing factors. The reactivity of pooled immune serum derived from broiler chickens immune to virulent C. perfringens to CP4, CP5, and CP6-derived secreted proteins was significantly different ^[42][145]. Additionally, the predisposing factors used in the disease models, such as pre-infection with Eimeria spp. and inclusion of fish meal in the diet, influence the severity of necrotic enteritis and the immune response elicited ^[43][146]. Other factors such as the timing of challenge, the use of a coccidial vaccine or virulent Eimeria spp., the dose of challenge, and research intention ^[44][147] will affect the immune response.