CD4+ Cell Development, Function & Dysregulation in Allergy and Autoimmune Diseases
CD4+ Cell Development, Function & Dysregulation in Allergy and Autoimmune Diseases
The helper T (TH) cells in the body are central to all branches of the immune system. They are very aggressive cells, due to their role as ‘molecular guardians of the body’ and so they must be regulated and controlled. If this regulation or self-tolerance breaks down, then this provides a window for the development of allergy, being a “disease following a response by the immune system to an otherwise innocuous antigen” , or autoimmunity, where the body’s immune system targets self antigens. Before an understanding of the role of different CD4+ cells in immunopathology can be achieved, an appreciation of the different characteristics and interactions of these cells must first be considered.
After preparation of antigen onto a major histocompatibility complex-II (MHC-II), an antigen presenting cell (APC), typically a dendritic cell (DC) or macrophage, will present this to a naive TH cell. Depending on what type of pathogen has been encountered, the APC will stimulate the polarisation of the naive cell to one subset of TH cells. This is achieved by the release of specific cytokines from the APC to the TH cell, and the subsets of CD4+ cells are each defined by their unique surface receptors, the cytokines they release and by the types of invaders they target.
The first subset to be discovered was TH1. These target intracellular pathogens, such as Mycobacterium spp. and mainly utilise phagocytic and cytotoxic cells. Alongside antigen presentation, interferon- γ (IFN-γ) and interleukin-12 (IL-12) are required from the APC. IL-12Rβ2, on the CD4+ cell, then induces phosphorylation of the signal transducer and activator of transcription 4 protein (STAT4) and T-bet activation. The cytokines produced by this cell type, due to STAT4 and T-bet are mostly pro-inflammatory, such as IFN-γ (which up-regulates T-bet thus forming a positive feedback loop), tumour necrosis factor β (TNFβ), IL-2 and lymphotoxin-α (LTα)[1-3].
Around this time, TH2 cells were also discovered in response to extracellular pathogens and parasites e.g. helminths, and these cells induce humoral immunity via immunoglobulin G4 (IgG4) and IgE production. IL-4 and IL-2 induce this subset, by IL-4R directed phosphorylation of STAT6 which, with GATA-sequence binding protein 3 (GATA3), causes transcription of IL-4. This is the positive feedback source as IL-4 up-regulates GATA3. The cytokine profile for this cell includes IL-4 and IL-13 (which induce IgE class switching), IL-5 and Amphiregulin (for eosinophil recruitment) and IL-25 (a self-stimulating cytokine)[1-5]. Note that polymorphisms in GATA3 and IL-4Rα (at position 576. glu→arg) have been associated with hypersecretion of IgE from B cells.
For reasons that shall be explained later, TH1 cells were associated with inflammatory autoimmunity, and TH2 cells with allergies and asthma. Very recently, a new subset called TH17 was discovered, which initiated rethinking of the TH1-TH2 mediation hypothesis. This type of cell is polarised in extracellular bacterial and fungal infections[2,3,6]. Like TH1, this type of cell produces very potent pro-inflammatory cytokines. TH17 lineage is first induced by low levels of transforming growth factor β (TGFβ), and IL-1β/IL-6. This up-regulates IL-23R (which is not present on naive CD4+ cells), sensitising the cells to IL-23, and causes cytokine production by RAR-related orphan receptor γt (RORγt) expression. These include IL-17, IL-17F, IL-21, IL-22 and TNFα. At this point, IL-21 provides the autocrine positive feedback system, causing proliferation. IL-21’s importance is highlighted by the condition XSCID, where individuals have no functioning IL-21R. As B cells have high levels of IL-21R, this results in no humoral responses to infection. To maintain the population of TH17, IL-23 is required, otherwise the cells dissipate[1,2,4,6,8].
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The final type of CD4+ cell to be considered is the CD4+CD25+ Treg cell, and this is due to the close relationship between TH17 and induced Treg (iTreg) cells. The main concern of these cells is that of self tolerance, and immunity regulation. They are induced by the presence of IL-2, high levels of TGFβ but absolutely no proinflammatory cytokine presence. Instead of TGFβ , all-trans retinoic acid (ATRA) or IL-10 can be used to induce this lineage. It can already be seen that there is a connection with TH17 cells by TGFβ, and because of this RORγt is also expressed in iTregs. However, via STAT5, the master regulatory gene, Forkhead box p3 (Foxp3), is also transcribed, and the Foxp3 protein antagonistically binds to the RORγt protein. These cells produce suppressive cytokines such as IL-10 and IL-35, and sometimes, by IL-10, iTregs can induce plasticity of different CD4+ subsets or production of IL-10 by TH cells, thus making them self-regulatory[2,6,9].
Treg cells are responsible for resisting hyperactivity of all TH cells, including TH1, TH2 and TH17. They employ the cytokines they produce and other techniques to do this, but the body has to be able to fight pathogens if actually present. Toll-like receptor (TLR) signalling is a good example of this, as such a signal suppresses Treg populations to allow TH activity. However, inability to restore homeostasis of the Treg/TH17 balance allows uncontrolled inflammation, potentially in autoimmunity, and/or lack of control of the other subsets, leading to their ‘signature’ immunopathologies.
Although all of these cells exist, and can individually proliferate as has been outlined, there are inhibitory mechanisms to this, and mostly they are intercellular from within the CD4+ population. TH1 and TH2 are antagonists, but they both prevent TH17 polarisation. Both IFN-γ and IL-4 prevent production of IL-23 which is absolutely necessary for TH17 stabilisation. They inhibit each other as T-bet suppresses GATA3, whereas GATA3 downregulates STAT4 and up-regulates STAT5. STAT5, in turn, inhibits T-bet, so each has a polarisation factor that prevents polarisation factors from other lineages being expressed. Also, lack of thymic stromal lymphopoietin (TSLP) allows TH1 dominance, whereas lack of interferon regulatory factor 2 (IRF2) or T-bet-/- had the opposite effect, directing towards TH2. Therefore, as TH1 and TH2 logically cannot be co-localised, IFN-γ and IL-4 control the pivot between cell mediation and humoral mediation[1,2,4,6,8].
In addition to this, TH17 is regulated by iTreg cells, but iTreg cells are regulated by TH17 also, due to the fact that they are induced by common factors, representing a physiological balance. To prevent the cell being directed to a TH17 cell, IL-2, via STAT5, causes Foxp3 expression, and the section of the protein encoded by exon 2 of the Foxp3 gene binds to RORγt inhibiting its action. Also, STAT5 competitively binds to IL-17 along with STAT3, preventing expression[2,6]. These cells, however, are joined in their inhibition of TH1 and TH2 cells, by TGFβ as this turns off GATA3, IL-4, and IL-5 among other genes. It also turns on IL-9, inducing TH2 to TH9 plasticity.
Treg cells have a high affinity IL-2R due to IL-2’s importance as a survival-growth factor. It abrogates IL-2 from the surrounding environment, and so very effectively inhibits TH1 and TH2 survival, but as IL-2 inhibits TH17, this could be a fault in its regulatory function against TH17. Tregs may also resort to plasticity as a form of regulation, as it has been recorded that supplying IL-23 to TH2/TH1 populations can induce TH17, or the opposite direction with an appropriate cytokine can also be achieved. This is very difficult to do, but may be viable due to the instability of iTregs and so the lack of their ability to monitor these cells at all times[2,6,8]. In X-linked immunodeficiency polyendocrinopathy enteropathy (IPEX), Foxp3 has a frameshift mutation, leading to fatal autoimmunity, usually by a TH2 hyperactivity related condition. As Foxp3 functionality is a non-redundant requirement for Tregs, this highlights the importance of TH regulation to avoid autoimmunity and/or allergy[2,6,9].
These pathways all reveal a very stringently controlled system, and this is for a reason. In the collapse, or bypass of this system, abnormal activity of CD4+ cells can have detrimental effects on the body, even to the point of fatality. Using the above example as a starting point, TH2 dysregulation typically is associated with allergy. Their production of IL-5 recruits eosinophils, and IL-4 and IL-13 induce class switching to IgE. These then bind to FcεRI located on mast cells and basophils, activating them. This is called sensitisation and upon exposure to the allergen the immunoglobulin recognises, these cells degranulate releasing histamine and heparin[10,11].
Alternative macrophages accompany in this condition, and can release more cytokines exasperating the allergic situation. Such cytokines include IL-25, IL-33, TSLP and IL-9. Recruited eosinophils produce IL-25, promoting TH2 function. This increases IgE levels and mucin production by goblet cell metaplasia for example. IL-33 acts in the same way. The allergenic role of IL-9 highlights the problem with dysfunctioning of Treg cells, as TH2 induction to TH9 has been mentioned as a mechanism used for alleviation of TH2 activity. IL-9R is present on basophils, and so their activation by IL-9 from TH9 cells results in the expansion of the TH2 population, and it also increases activity of eosinophils, due to the cytokines that activated basophils release[8,10].
A further example of how lack of regulation can lead to a TH2 skew is the presence or absence of Vitamin D3 and IL-10. Although producible by a great number of cells, both nTreg and iTreg cells are prime sources of IL-10, and in many cases are responsible for the induction of IL-10 production in TH1 and TH2 cells, so causing self-regulation. IL-10 is key to reducing IgE levels, and Vitamin D3 is required for Treg sustaining and so IL-10 maintenance. Consistent exposure to the common particles (allergens) is required for the Treg population to maintain the specific IL-10 production. A drop in this can lead to a drop in Tregs and so a rise in TH2. In fact, even with Treg cells, polymorphic IL-10 promoters have been highlighted in the reduction of IL-10 mRNA and increase in proinflammatory mRNA in allergy.
However, despite this obvious role of TH2 cells in allergies, and particularly in asthma, more cells direct the immunopathology than just TH2. For example, TH17 cytokines, such as IL-17, can be detected in metal allergies, atopic dermatitis and also in the sputum of non-eosinophilic asthma patients, along with neutrophils. When only TH2 cells are detected, elevated eosinophil levels are present; with TH17 only asthma, elevated neutrophils can be found but with both TH2 and TH17 cells present, extreme eosinophilia sets in. This seems illogical, as the co-presence allegedly promotes TH2 function. Also, the method used to sensitise the patient seems to be important, as Langerhans cells in an ovalbumin sensitised (OVA) mouse model induce TH17 cells. The resulting correlation between TH17 cytokines and neutrophilic presence and activity reveal a prominent role of TH17 in this particular asthma activation pathway.
The combination of antigen presentation with TSLP can be used to initiate TH2 activation, and so this would also be usable in allergies. In an allergic situation, such as allergic asthma, if this is combined with dsRNA (highlighting viral presence) a very strong polarisation to TH2 and TH17 occurs. This therefore induces a very potent inflammatory response to this infection, and so viruses can be seen to aggravate such conditions. TH2 cells but also TH1 and TH17 cells, particularly in inflammatory allergic conditions, have all been detected and associated with this regulatory breakdown. This reinforces the statement that allergic conditions are not down to one cell’s dysregulation, but that a complex involvement of many cell subsets is present.
It has been hypothesised that there could be plasticity from TH17 to TH2, as only half of the original TH17 cells in some studies produce TH17 cytokines in the allergic patient, and also chronic progression through various cells over the course of the condition is plausible[3,11]. Therefore, targeting purely the TH2 cells may not prove effective, e.g. via corticosteroids, due to the presence and proliferation (that would result from a drop in TH2) of other, inflammatory cells. The inclusion of Vitamin D3 with the allergen doses in immunotherapy seems to be promising, as this could require less allergen to be provided, lessening the risk of anaphylaxis, but also promoting IL-10 production and TH suppression or polarisation away from TH2.
Hypersensitivity doesn’t only apply to the immediate form, as in allergy, but also immune cells may be directed against self antigens, as is the case in autoimmunity. Originally, TH1 cells were designated the main protagonists, owing to their persistent presence with cytokines in experimental autoimmune encephalitis (EAE), rheumatoid arthritis, Crohn’s disease and psoriasis sites. However, IL-12-/- genotypes provided no resistance to the conditions. In fact it made them worse. IL-12 is heterodimeric, composed of p35 and p40 subunits. IL-23 shares the p40 subunit, combined with p19, to make its structure. IFN-γ-/- and p35-/- had the same effect as total loss of IL-12, but p40-/-, p19-/- and IL-23-/- all conferred resistance against these pro-inflammatory autoimmune conditions[1,3].
The focus on genes here is significant, due the strong role genetics has regarding autoimmune susceptibility. For example, dizygotic twins have a 5-6% chance of developing the same autoimmune disease. However, a pair of monozygotic twins have a 30-50% chance of having the same autoimmune hypersensitivities.
TH17 cells are very potent in autoimmune inflammation, however the presence of TH1 cells does signify at least some involvement, potentially in continuation. The co-expression of IFN-γ and IL-23 in chronic conditions further exemplifies this, but even more accurate is the rise in TH1 cytokines after the fall in the instigating IL-17, promoting a hypothesis of complex systematic processes that go through cellular phases[4,5]. In various types of autoimmunity, auto-antibodies are seen to be involved, highlighting a potential role of TH2 cells, but consistently the employment of inflammatory and cytotoxic cells by TH1 and TH17 cells is a signature feature.
The positive feedback loop between prostaglandin E2 (PGE2) from macrophages and IL-17 from TH17 cells causes the proliferation and maintenance of potent TH17 cytokines, including up-regulated IL-1β, IL-6, IL-17 and IL-22. In particular, IL-17 and IL-17F are seen to be non-redundant in autoimmunity. Not only do the TH17 cells produce these cytokines, but in autoimmunity the production of pro-inflammatory cytokines from other cells is induced, such as IL-23 and TNFα from Paneth cells in Crohn’s Disease and Ankylosing Spondylitis. This is possibly due to up-regulated IL-23R in these conditions. The massive amounts of these cytokines being produced, and the fact that it encourages a positive feedback system, employing more macrophages, neutrophils and TH17 cells reinforces the importance of TH17 cytokines in autoimmunity[4,5].
Due to the expression of Aryl hydrocarbon receptor (AhR) on TH17, the binding of aromatics, such as carbazole from cigarette smoke, can cause the exasperation of an already serious autoimmune condition. LPS can also aggravate autoimmune conditions, such as in Crohn’s disease, as the secretion of pro-inflammatory IL-22 results from TLR activation by LPS[5,6].
High levels of iTreg cells are detectible in autoimmunity, but they are unable to quell the proliferation and activity of the inflammatory TH1 and TH17 cells, despite releasing high levels of SOCS3 which inhibits IL-23. This is due to overwhelming levels of IL-6, TNFα and DC activation of cells. Even complete removal of TGFβ doesn’t provide resistance, as although TH17 cells are no longer present, there is now a lack of Tregs. This allows severe hyperactivity of both TH1 and TH2 cells, resulting in 100% mortality in mouse models[1,5,6].
What is obvious from the study of this topic is that TH cell dysregulation is not solely responsible for autoimmunity and allergy. Of course the breakdown of tolerance, and regulation are severally detrimental, but genetic susceptibility regarding MHC phenotypes, such as HLA-DR3 and HLA-DR4 in type 1 diabetes mellitus, must also be considered. Beyond this, environmental factors outside of the body’s control, innate cell activity, APC activity, the characteristics of the cells to which the allergic or autoimmune condition is localised, and many other factors all play a role in the development of hypersensitive conditions.
The only true inference that can be taken from this information is that despite strong associations of certain cells with specific conditions, no one single cell is responsible for a condition. TH2 are accompanied by TH17 in allergies, and TH17 by TH1 and TH2 in autoimmunities, just showing how the only common feature throughout all of these conditions is that they all involve immensely complex and potent processes, many cells and no simple answers.
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