Extra-adrenal glucocorticoid biosynthesis: implications for autoimmune and inflammatory disorders.

Multiple sclerosis (MS) is a chronic autoimmune disease that affects the central nervous system 99 , 100 . Multiple sclerosis affects about 1 in 400 adults with women being twice as likely to be affected by the disease than men 101 , 102 . One likely mechanism giving rise to multiple sclerosis is that the overactive T helper cells (Th1 and Th17 cells) promote inflammation that results in demyelination 103 . The demyelination leads to the damage of the blood brain barrier (BBB), thus resulting in immune cells such as macrophages, T cells, and B cells infiltrating the brain and causing further inflammation and the eventual formation of scar tissue 103 , 104 . The main types of multiple sclerosis are relapsing/remitting MS (RMMS), secondary-progressive MS (SPMS), primary progressive MS (PPMS), and progressive- relapsing MS 103 , 105 , 106 . Progressive- relapsing MS used to be a subtype, but in 2013 was removed due to it being considered as repetitive 105 . RMMS is the most common type of MS representing about 85% of MS cases, followed by PPMS which represents 8–10% of MS cases 103 . Systemic lupus erythematosus (SLE) is an autoimmune disease that presents with multiple symptoms; however, the effects of the disease seem to result from the formation and deposition of autoantibodies 107 – 109 . The causes of the disease remain to be further investigated; however, there are multiple etiological factors (genetic, immunological, hormonal, etc) involved in the disease 107 , 110 . The disease seems to be related to the dysregulation of the Th2 cells as there is an increase in Th2 cytokines (IL-4, IL-5, and IL-13) in SLE patients 108 . SLE also happens to affect females more than men 110 , 111 . Fortunately, because of better treatment the survival rate for SLE has improved significantly from a 5% survival rate over a 5 year period in the 1950s to a 99% survival rate over a 5 year period in the 2010s 111 . Rheumatoid arthritis (RA) is an autoimmune disease that is present in 1% of the population 112 . This disease adversely affects the quality of life and productivity of the patient, and there are high costs for therapy and its monitoring for toxicity 113 . RA is characterized by chronic synovitis, which shows a predilection for diarthrodial joints, particularly the metacarpophalangeal (MCP) and proximal interphalangeal joints (PIP joints) 112 . A preponderance of evidence indicates that an antigen-driven immune process against one or more proteins found in cartilage sustains synovial inflammation in RA 114 , 115 . Each DMARD and biologic used to treat RA has the potential to cause unique serious adverse events and morbidities or mortalities. These include lung fibrosis, fulminant infections, inflammatory demyelination, liver cirrhosis, development of skin cancer or melanoma, retinal damage and triggering the onset of other autoimmune diseases such as vasculitis, MS and SLE 112 , 116 – 120 . Psoriasis is a chronic inflammatory disorder of the skin affecting 1-9% of the population 121 – 123 . The etiology of this diseases is multifactorial with a crucial role assigned to the malfunctioning of the immune system with the dysregulation of T cells, particularly of the Th1 and Th17 lineages 121 , 123 , 124 . Although the exact mechanisms need further investigation, it is accepted that the disease progression is driven by cytokines, particularly IL-17 and IL-23 123 , 124 . In addition, stress and deregulation of local and systemic neuroendocrine functions have been implicated in the etiology and natural history of psoriasis 71 , 125 – 127 . Currently, the main treatment for mild psoriasis encompasses the use of corticosteroids with or without additional topical vitamin D derivatives 128 . Ultraviolet light therapy is also used to treat psoriasis 129 . More recently, biologics that can target the IL-23/IL-17 pathways as well as JAK inhibitors that target IL-12 and IL-23 cytokines have been used in the therapy of this disease 130 , 131 . Atopic dermatitis is the most common skin inflammatory disease, affecting millions of people 132 . Like psoriasis the exact mechanism of this disease needs to be further investigated, although some of its causes include dysregulation of filaggrin and epidermal barrier function, and dysregulation of Th1 and Th2 effector cells 132 , 133 . The acute phase of atopic dermatitis is mediated by Th2, while the chronic phase is mediated by Th1 cells 133 . Treatments for atopic dermatitis include the use of corticosteroids, cyclosporine, and more recently biologics that target IL-5 and IL-13, and potentially JAK inhibitors 131 .

Glucocorticoids play many roles in the maintenance of homeostasis in the body including displaying important immunosuppressive activity. Glucocorticoid synthesis is regulated by ACTH, CRH, and cytokines such as IL-1, IL-6 and TNFα in a context-dependent fashion. While some of the regulators such as ACTH directly display immunosuppressive effects, others such as CRH and cytokines have predominantly pro-inflammatory activity. Therefore, in peripheral organs dissociation of the actions of the higher-level regulators, CRH and proinflammatory cytokines, from the executive arm involving the synthesis of glucocorticoids, can lead to uncontrolled stimulation of the immune system. Furthermore, our view is that dysregulation of local (immune cells and or target organs for immune activity) glucocorticoid synthesis plays a pivotal role in several autoimmune diseases, including MS, LE, and RA, as well as proinflammatory skin diseases such as psoriasis and AD ( Figure 5 ). Creative investigations on how to pharmacologically target local and endogenous glucocorticoid biosynthesis and glucocorticoid signaling should help to find future therapies/cures for inflammatory and autoimmune diseases. In particular, there needs to be targeted research aimed at increasing local cortisol/corticosterone levels through the activation of their local synthesis without systemic effects and/or by preventing their inactivation, and/or by stimulation of the activity of 11βHSD1/2. The precise delivery of factors regulating glucocorticoid biosynthesis to the target organs or immune cells should also be a focus of future research. Such agents that are able to directly or indirectly influence local cortisol levels may be chemically synthesized in an educated fashion or represent natural products identified by medicinal chemistry and computer modeling. In addition, the application of different types of physical factors such as ultraviolet B (UVB) radiation in a controlled fashion may represent an additional opportunity, since UVB is both immunosuppressive and also stimulates glucocorticoid biosynthesis. In conclusion, local cortisol levels can influence the development or regression of inflammatory (psoriasis, AD) and autoimmune diseases such as LE, MS and RA. Research aimed at modulating local levels of cortisol is necessary to provide new therapies to patients suffering from these devastating diseases.

Peripheral CRH can be synthesized by cells of the immune system, somatic cells and by peripheral afferent type sensory fibers and postganglionic sympathetic nerves 134 , 135 . Many tissues (e.g. skin, synovium of patients with RA, colonic mucosa of patients with ulcerative colitis, ovaries, cardiovascular system, eyes, uterus, adipose tissue, thymus, bladder, liver, stomach and kidney) express CRH and/or CRH receptors (CRHR) 64 , 136 – 145 . The most extensively studied site has been the skin of humans and mice which revealed the presence of not only CRH and CRH-R1/2, but also peptides derived from POMC 64 . Whether an HPA-like axis is present and operative in synovium, gastrointestinal tract or other extracranial locations requires further studies. Although some authors reported expression of a truncated POMC mRNA in fibroblasts from human synovium, they failed to detect POMC protein in osteoarthritis synovial tissue and proper melanocortin signaling 146 . However, latter studies by these authors indicated a role for POMC signaling including MSH and ACTH in osteoarticular tissues with anti-inflammatory actions 147 , 148 . Also, truncated POMC mRNA can be translated into full-length POMC protein that is processed to the corresponding downstream neuropeptides 149 – 151 . The POMC-derived peptides, ACTH and β-endorphin (β-End), are expressed in the synovium of RA patients and are produced by lipopolysaccharide stimulated B lymphocytes 152 . Analysis by double immunostaining of arthritic synovial tissue from Lewis rats with adjuvant arthritis showed that both ACTH and CRH colocalized in fibroblast-like cells and in mononuclear cells 152 . Various stimulants or stressors including phytohemagglutinin, concanavalin A, and IL-2, induce lymphocytes to express CRH mRNA and/or CRH protein 153 . Human monocyte- derived dendritic cells produce CRH mRNA and protein when stimulated by the intestinal commensal bacteria bacteroides vulgatus and fusobacterium varium 154 . Production of PGE2 by explants of RA synovial tissue is increased in the presence of CRH 155 . Locally produced CRH modulates both pro-inflammatory and anti-inflammatory processes. This is supported by its ability to stimulate production of IL-6 from blood monocytes, increase leukocyte IL-1 and IL-2 secretion, suppress LPS- induced IL-1 and IL-6 by peripheral blood mononuclear cells, stimulate lymphocyte expansion, and IL-2 receptor expression, inhibit splenocyte proliferation induced by IL-2, facilitate NK cell mediated cell lysis; and stimulate the of production of ACTH and B-END by leukocytes 156 – 161 . CRH induces macrophages and mast cells to produce and release VEGF, IL-1β and IL-6 84 and human peripheral blood CD14 + monocytes to produce TNFα and dysfunction of vascular endothelium 162 . CRH upregulates IL-4 production by human Th2 T cells, downregulates IFN-ɣ production by human Th1 T cells and downregulates IL-10 production by FoxP3- negative human peripheral blood T regulatory cells 163 . The different effects of CRH on inflammation and immune function may be influenced by different CRH receptors being expressed on different types of leukocytes or by somatic cells in different tissues 84 . The effects of CRH are mediated via two different receptors, CRH-R1 and CRH-R2, which are members of class B1 of G-protein coupled receptors that exhibit approximately 70% overall homology at the amino acid sequence level but only about 47% homology in the N-terminal extracellular domain 164 . CRH binds to CRH-R1 with greater affinity than it binds to CRH-R2 165 . Alternative splicing gives rise to at least 8 spliced variants of CRH-R1 and at least 3 spliced variants of CRH-R2 84 , 85 , 166 . Both the pro-inflammatory and anti-inflammatory effects have been reported to be mediated by either CRH-R1 or CRH-R2 indicating that the ultimate effect of signaling via these receptors is determined by factors other than the specific type of CRH-R. For example, CRH via CRH-R1 induces mast cells and macrophages to produce IL-6, IL-1β, TNFα and VEGF and promotes vasculitis but has also been shown to block IL-1α-stimulated prostaglandin synthesis by fibroblasts 84 . Similarly, in the early stages of inflammation, CRH via CRH-R2, suppresses production of TNFα by macrophages activated by LPS, but has the opposite effect on LPS-induced macrophage TNFα production in late stage inflammation 167 . Since the initial detection of CRH, CRH-R1, and CRH-R2 in human 168 – 173 and murine 168 , 174 – 176 skin, a flurry of reports documented their regulated expression in the mammalian skin (reviewed in 71 , 79 , 177 – 180 ). CRH and urocortins acting on cutaneous CRH-R1 and CRH-R2 can affect skin functions in a context-dependent fashion 71 , 75 , 79 , 181 , 182 . The direct CRH effects are predominantly anti-proliferative, pro-differentiation, barrier building and pro-inflammatory. However, indirect effects through activation of POMC or glucocorticoid signaling can be anti-inflammatory 71 , 79 , 80 (see below). It should be noted that since the original discoveries on POMC expression and production of POMC peptides by skin cells 149 , 183 – 185 , it has been widely established that skin cells can produce different POMC peptides in a context dependent fashion under different stimuli to regulate different skin functions, including downregulation of pro-inflammatory responses 64 , 186 . 129 , 178

The dysregulation of skin steroidogenesis may play a role in both psoriasis and atopic dermatitis 188 , 231 , 232 . Glucocorticoids act by blocking the production of IL-4 and IL-5 231 , 233 . Hannen et al. reported that in the skin, the expression of several enzymes involved in steroid synthesis such as CYP11A1 and CYP17 are reduced in psoriasis, as well as the enzymes 11βHSD1, 11βHSD2, and the GR 128 . They further demonstrated that StAR and MLN64 expression is reduced in skin of both atopic dermatitis and psoriasis patients 193 . Tiala et al. reported that CCHCR1, a gene that plays a role in steroidogenesis and vitamin D metabolism, is downregulated in psoriasis 234 . Another study reported that deficient in situ synthesis of glucocorticoids in psoriatic skin was associated with increased inflammation 235 . The above data support the hypothesis that defective glucocorticoid signaling contributes to the pathogenesis of psoriasis 206 . Local steroidogenesis might also play a role in the prevention of multiple sclerosis. Boghozian et al. found lower CYP17A1 expression levels as well as lower dehydroepiandrosterone (DHEA) levels in oligodendrocytes in both MS patients and animals with EAE (experimental autoimmune encephalomyelitis) 236 . This group also found increased expression of IL-1β and IFN-γ in MS patients. The results seem to suggest that DHEA may play a role in immunoregulation. Arnason et al. reported that ACTH can be beneficial for MS patients, although Miller et al found ACTH to be a candidate for the therapy of multiple sclerosis as early as 1961 237 , 238 . Arnason et al. later described how melanocortins are anti-inflammatory and act by blocking NF-kB , and that melanocortins exert their anti-inflammatory effects through melanocortin receptors MC1R, MC3R, and MC5R 237 . The expression of genes encoding certain enzymes producing sex hormones as well as the receptors for these hormones may also be implicated in the pathogenesis of multiple sclerosis 101 . For example, Luchetti et al. reported that the MS lesions in males display higher expression of mRNA for aromatase, estrogen receptor B, and TNF, while women with MS have increased expression of mRNA for 3β-hydroxysteroid dehydrogenase and the progesterone receptor 101 . The dysregulation of steroidogenesis could be a contributing factor to the pathogenesis of SLE. Corticosteroids are used in first line treatment of patients with SLE 108 , 188 , 239 . Glucocorticoids affect T cells (especially CD4) more rapidly than B cells 239 . Glucocorticoids affect the T cells by enhancing circulatory emigration, inducing apoptosis, inhibiting T cell growth factors, and impairing the release of cells from lymphoid tissue 239 . ACTH has been used since the 1950s as a treatment option for SLE 240 , 241 . Vogl et al. have found that a number of steroids are lower in SLE patients than control patients, specifically progesterone, 17-hydropregnenolone, and cortisol 242 . Li et al compared pituitary hormone level in SLE patients verses the healthy controls and found that prolactin levels were increased in SLE patients 110 . Steroidogenesis as well as the factors that regulate it may play an important role in the pathogenesis of rheumatoid arthritis. This is not surprising since 100–2000 genes are regulated by glucocorticoids 243 . Yoursi et al found that there are 32 steroid-like metabolites whose concentration differ significantly between RA patients and healthy controls 244 . These metabolites included DHEA, adrostenediol, and cortisol 244 . Straub et al reported that serum levels of cortisol, DHEA, and DHEA-S levels were elevated in early rheumatoid arthritis patients compared to healthy individuals and correlated with elevated levels of the proinflammatory cytokines, IL-6 and TNF 245 . This group speculated that this might be due to RA patients having a deficiency in either CYP21A2 or CYP11A1 245 . In another report, Straub et al noted that the relatively low levels of steroids in RA patients in relation to proinflammatory cytokines was not due to increased renal clearance, and in fact the renal clearance of steroids, including androgens, was decreased in RA patients 246 . Schlaghecke et al found that the PBMCs (peripheral blood mononuclear cells) in RA patients have a lower density of glucocorticoid receptors that healthy controls 247 . However, Schlaghecke later reported that this decreased GC density does not cause glucocorticoid resistance in RA patients 248 . In a review article about the role of 11βHSD1 and 2 in RA, Edwards concluded that overactivity of 11βHSD1 can cause dysregulation of the HPA controlling cortisol production 249 . He also speculated that the proinflammatory cytokine, TNFα ,triggers the overactivity of 11βHSD1 and that anti-TNFα therapy can be beneficial in RA 249 . Finding out the exact mechanism by which steroidogenesis is dysregulated in RA may open doors for discovering new treatment options in RA. Specifically, the regulation of the local interconversion of cortisol and cortisone and/or glucocorticoid biosynthesis and CYP11A1 activity may be targeted in immune cells or their target organs.

The biosynthesis of steroid hormones starts from cholesterol, which in turn is derived from a number of sources including de novo synthesis, lipoprotein-derived cholesteryl esters, and cholesteryl esters stored in lipid droplets 1 – 3 . Hormonal regulation of steroid biosynthesis occurs within minutes (acute) to hours (chronic) and is primarily mediated by cAMP signaling 3 – 7 . Steroid hormones are largely synthesized in steroidogenic cells of the adrenal, ovary, testis, placenta, and brain; however, they are also produced in a number of extra-adrenal and -gonadal tissues. Glucocorticoids play critical roles in a wide variety of physiological processes, including regulation of various developmental and homeostatic pathways and display several immune functions 3 , 8 . Their release and production are regulated primarily by ACTH (adrenocorticotropic hormone) and indirectly by CRH (corticotropin releasing hormone) 1 . Autoimmune disease and skin inflammatory disorders represent a significant clinical problem affecting large segments of the population and the quality of life of affected patients and impose a significant cost to the economy, and the health care system in particular. While there are different factors underlying the etiology of multiple sclerosis (MS), lupus erythematosus (LE), rheumatoid arthritis (RA) and skin inflammatory disorders such as psoriasis and atopic dermatitis (AD), they are linked by one element, the diseases are a consequence of a dysfunctional/hyperactive immune system. Glucocorticoids are used worldwide to treat autoimmune disease and inflammatory disorders. Since the skin and systemic immune cells can produce glucocorticoids as well as their hormonal regulators, we are exploring the hypothesis that autoimmune and inflammatory diseases develop and progress due to a malfunction of local glucocorticosteroid signaling and that their regulators play a role in the development and progression of autoimmune and inflammatory diseases.

It has been reported that glucocorticoids can be synthesized in many non-adrenal and non-gonadal tissues, such as the brain, intestine, lung, skin, spleen, placenta, adipose tissue and the immune system, as well as in a variety of cancer cells 42 , 52 , 187 – 189 . Table 1 shows the distribution of the proteins involved in the initial rate-controlling steps of steroidogenesis, CYP11A1 and StAR, as well as other downstream steroidogenic enzymes reported to be in these tissues including immune cells ( Figure 3 ), at least at the level of mRNA expression. Some of the tissues listed such as bone, endometrium and mammary gland appear to primarily produce sex steroids and the production of corticosteroids from cholesterol remains to be established The skin has been shown to express all the CYP enzymes involved in steroid synthesis, including CYP11A1, CYP17A1, and CYP21A2 and StAR protein in both keratinocytes and sebaceous glands 188 , 190 – 193 ( Table 1 ; Figure 4 ). Moreover, the skin has also been shown to express CRH and POMC 170 . The incubation of melanocytes with CRH caused the melanocytes to produce ACTH , and in turn ACTH stimulated the production of cortisol in melanocytes 78 . Similarly, fibroblasts can produce cortisol as shown by liquid chromatography/ mass spectrometry (LC/MS) 194 , and production of corticosterone can be stimulated by CRH and ACTH. Finally, the exposure of dermal fibroblasts to CRH stimulates POMC activity and corticosterone production 77 , with UVB activating cutaneous elements of the HPA 195 – 198 . Thus, there is evidence that a functional peripheral HPA-like axis is operative in the skin 199 , 200 Glucocorticoid production in the skin is regulated by CRH, ACTH, IL-1β, UV light, and by 11βHSD1 and 11βHSD2 enzymes 52 , 188 , 192 . Stress to the skin either by inflammation or injury causes the stimulation of ACTH and POMC production in the skin 64 . UVB exposure of the skin has been found to cause production of CRH, ACTH, β-END, and cortisol 195 . The corticosteroids produced in the skin appear to play a role in countering the inflammatory response of the skin 192 , 201 . However, glucocorticoids produced locally can have a negative effect on barrier function and would healing and promote skin infection 202 – 206 . The thymus, a place where T lymphocyte maturation occurs, has been found to produce glucocorticoids 187 (see Table 1 ). In fact, de novo synthesis of steroids in the thymus was discovered in the mid 1990s by Vacchio et al. 207 . Vacchio also demonstrated the presence of the steroidogenic enzymes CYP11A1 and CYP11B1 by immunohistochemistry. In addition, thymic epithelial cells produced pregnenolone and deoxycorticosterone. Peripheral T cells have also been reported to produce steroids, particularly pregnenolone 208 . Importantly, there are recent reports showing the expression of CYP11A1 in human 209 and murine 208 , 210 , 211 T cells. We have also observed the expression of CYP11A1 in CD4 and CD8 human T lymphocyte and non-T cells (B cells and monocytes) as shown in Figure 3 . Inhibition of the expression of pro-inflammatory cytokines and synthesis by glucocorticoid is mediated by the binding of the glucocorticoid-GR complex to GREs in the promoter regions of these genes (e.g. IL-1α and IL-1β). This can block the binding and function of other transcription factors (e.g. nuclear factor-kappa-B (NF-κB), and activator protein-1(AP-1)) essential for transcriptional activation of proinflammatory mediators 212 – 216 . Inversely, NF-kB can also inhibit the function of the GR in a dose-dependent manner 217 indicating that these pathways mutually affect each other. The protein glucocorticoid-induced leucine zipper (GILZ) has been found to play a role in some of the anti-inflammatory effects of glucocorticoids 218 – 220 . These effects include, but are not limited to inhibiting NF-κB, Ras/Raf, and AP-1 dependent pathways 218 , 220 , 221 . Another mechanism for glucocorticoid-GCR mediated inhibition of inflammation is the recruitment of other transcription factors to promoter sequences of genes that code for proteins with anti-inflammatory properties (e.g. IL-10, NF-κB, IL-1 RII, GILZ, lipocortin-1, alpha-2-macroglobulin, and secretory leukocyte-protease inhibitor) 212 – 214 , 220 , 221 . Glucocorticoids can also mediate their anti-inflammatory effects at the post-translational level by decreasing the stability of mRNAs for IL-1,IL-2, IL-6, IL-8, TNFα and GMCSF, or increasing the stability of a number of other mRNAs. The latter include mRNAs for various enzymes (e.g. angiotensin - converting enzyme and neutral – endopeptidase) that degrade the vasodiliatory peptide (e.g. bradykinin), annexin-1( lipocortin-1, macrocortin and/or lipomodulin ) which has anti-inflammatory action by inhibiting phospholipase A2 leading to reduced generation of arachidonic acid from membrane phospholipids, and by decreasing the stability of cyclooxygenase-2 mRNA resulting in reduced production of PGE2 222 The global effects of glucocorticoids on leukocytes and endothelial cells lead to a decrease in the adherence of leukocytes to the endothelium of blood vessels which reduces their extravasation into areas of inflammation, thus reducing the inflammatory response 223 – 225 . Proliferation of B cells and T cells is inhibited as well as the production of immunoglobulins (B cells) and Th1 and Th2 cytokines (T cells). There is less inhibition of Th2 production than Th1 and there is attenuation of natural killer (NK) cell activation 177 , 226 , 227 . Glucocorticoids act on eosinophils to increase their apoptosis directly, or via reducing the production of IL-5 228 . Mast cell degranulation, cytokine production and their adherence to the endothelium are inhibited by glucocorticoids 177 , 229 . Glucocorticoids reduce the number of circulating monocytes and cause activation of antigen presentation functions of monocytes/macrophages/dendritic cells 177 , 230

The glucocorticoid receptor (GR; NR3C1), a member of the nuclear receptor superfamily, mediates the action of glucocorticoids. It contains 4 domains: an N-terminal transactivation domain (NTD), central DNA-binding domain (DBD), a C-terminal ligand binding domain (LBD), and a hinge domain that connects the DBD with the LBD 52 , 94 – 96 . There are two signaling pathways for GR: classical and non-classical 94 , 97 , 98 . GR is localized to the cytoplasm in association with a chaperone complex. In the classical GR signaling pathway, interaction with activating ligands induces a conformational change in GR and dissociation from the chaperone complex. GR subsequently translocates into the nucleus where it binds to GRE (glucocorticoid-responsive elements) and regulates the transcription of target genes 94 , 97 . The non-classical GR signaling pathway is characterized by rapid signaling which is transcription independent, and is dependent on various types of kinases 98 .

The HPA axis is the main regulator of the stress response as well as for systemic glucocorticoid production 61 , 62 . CRH is the key regulator of the HPA and is produced in the paraventricular nucleus (PVN) of the hypothalamus 63 . Under stress it is released to the hypophysial portal vessels and after entering the anterior pituitary gland, it binds to the CRH receptor type 1 (CRH-R1) on the corticotrophs. Here it stimulates the expression, synthesis, and processing of proopiomelanocortin (POMC) including production and release of ACTH from corticotrophs 61 – 64 . After entering the circulation, ACTH binds to the G-protein coupled 7-transmemebrane receptor, MC-2 (melanocortin type 2 receptor), in the zona fasciculata of the adrenal cortex. Then, via cAMP dependent mechanisms it stimulates the transport of cholesterol into the mitochondria and the synthesis and activity of steroidogenic enzymes resulting in increased production and secretion of cortisol and corticosterone 65 , 66 . Glucocorticoids inhibit POMC expression, ACTH secretion and production of CRH in a negative feedback loop 67 . Figure 2 shows a scheme for the regulation of the HPA axis. In the central HPA axis, various stressors cause the release of CRH from the hypothalamus which then indirectly regulates immune and inflammatory reactions through secretion of ACTH from the pituitary which subsequently stimulates glucocorticoid secretion by the adrenal glands 68 . Inflammatory cytokines, including IL-1, TNFα and IL-6, stimulate the hypothalamus to secrete CRH 69 . CRH is also produced in various peripheral tissues including immune cells 70 , skin 71 , and other organs 72 , 73 . The net effect of central CRH is immunosuppressive through activation of the HPA axis, while the direct effect of locally produced CRH is pro-inflammatory 70 , 71 , 74 – 76 . However, indirect immunosuppressive effects through stimulation of local production of POMC peptides and glucocorticoids are possible 77 – 80 . CRH, in addition to acting on CRH-R1 also acts on CRH-R2, and both receptors are widely distributed in the body 81 , 82 including skin 71 , 83 and the immune system 84 . CRH receptors are coupled to different second messengers including cAMP, IP3 (inositol triphosphate), and Ca 2+ 71 , 81 . There are different alternatively spliced isoforms of CRH-R1 and CRH-R2 with different functions 71 , 79 , 81 , 85 , 86 . CRH related peptides including urocortin 1–3 are also produced centrally and peripherally and these show different affinities for CRH-R1 and CRH-R2 87 , 88 . ACTH is synthesized as a part of the ~30 kD POMC precursor that undergoes cell-specific post translational processing by protein convertase 1 (PC1) to cleave the 39 amino acid (aa) ACTH peptide, as well as other neuropeptide precursors that are further processed by PC2 to melanocyte stimulating hormone-γ (γ-MSH), β-MSH and β-endorphin peptides 64 , 89 . ACTH can also be cleaved by PC2 and further processed to produce the 13aa α-MSH peptide. ACTH interacts not only with MC-2 as an exclusive ligand for this receptor, but also with other MC receptors (MC1, MC3–5) to regulate different functions including melanogenesis (via MC-1) 90 – 92 . ACTH can also act directly as an immunosuppressor 64 , 93 .