Autocrine vitamin D signaling switches off pro-inflammatory programs of TH1 cells
Abstract
The molecular mechanisms governing orderly shutdown and retraction of CD4+ type 1 helper T (TH1) cell responses remain poorly understood. Here we show that complement triggers contraction of TH1 responses by inducing intrinsic expression of the vitamin D (VitD) receptor and the VitD-activating enzyme CYP27B1, permitting T cells to both activate and respond to VitD. VitD then initiated the transition from pro-inflammatory interferon-γ+ TH1 cells to suppressive interleukin-10+ cells. This process was primed by dynamic changes in the epigenetic landscape of CD4+ T cells, generating super-enhancers and recruiting several transcription factors, notably c-JUN, STAT3 and BACH2, which together with VitD receptor shaped the transcriptional response to VitD. Accordingly, VitD did not induce interleukin-10 expression in cells with dysfunctional BACH2 or STAT3. Bronchoalveolar lavage fluid CD4+ T cells of patients with COVID-19 were TH1-skewed and showed de-repression of genes downregulated by VitD, from either lack of substrate (VitD deficiency) and/or abnormal regulation of this system.
Discussion
We showed that cell-intrinsic complement orchestrates an autocrine/paracrine autoregulatory VitD loop to initiate TH1 shutdown. VitD causes genome-wide epigenetic remodeling, induces and recruits TFs, including STAT3, c-JUN and BACH2, which collectively repress TH1 and TH17 programs and induce IL-10 via IL-6–STAT3 signaling. This program is abnormal in lung helper T cells of patients with severe COVID-19, which show preferential TH1 skewing and could be potentially exploited therapeutically by using VitD as an adjunct treatment.
IFN-γ-producing airway helper T cells are key components of immunity to coronaviruses, including SARS-CoV1 and MERS-CoV20. TH1-polarized responses are also a feature of SARS-CoV2 in humans24 and severe COVID-19 is accompanied by prolonged, exacerbated, circulating TH1 responses21. Complement receptor signaling is a driver of TH1 differentiation and required for effective antiviral responses12,42. C3 cleavage generates C3b, which binds CD46 on T cells. We have previously shown that the lungs in COVID-19 are a complement-rich microenvironment, that local CD4+ T lymphocytes have a CD46-activated signature13 and show here that these T cells are TH1-polarized. Pro-inflammatory function is important for pathogen clearance, but a switch into IL-10 production is a natural component during successful transition into the TH1 shutdown program and reduces collateral damage16. Inability to produce IL-10 results in more efficient clearance of infections but severe tissue damage from uncontrolled TH1 responses results in death2. The benefits of remediating inflammatory pathways in severe COVID-19 is demonstrated by successful trials of dexamethasone, an immunosuppressive drug that reduces mortality1.
VitD has pleiotropic functions in the immune system, including antimicrobial as well as regulatory properties, which are cell- and context-dependent43,44. VitD deficiency is associated with higher prevalence and worse outcomes from infections, including influenza, tuberculosis and viral upper respiratory tract illnesses17, as well as autoimmune diseases, including type 1 diabetes, multiple sclerosis, rheumatoid arthritis and inflammatory bowel disease18. Helper T cells play key roles in all these diseases. Thus, understanding VitD biology in helper T cells has potential translational impact.
Our data indicate the complexities of TFs working within networks to regulate sets of genes. After VitD ligates VDR, c-JUN, STAT3 and BACH2 are recruited to acetylated loci, shaping the transcriptional response to VitD. c-JUN is a member of the AP-1 basic leucine zipper family, primarily involved in DNA transcription45. This TF was bound adjacent to 40% of VitD-regulated coding loci. BACH2 is a critical immunoregulatory TF38,39 downregulated in lesional versus non-lesional psoriatic skin46. Active VitD concentrations inversely correlate with severity of psoriasis40. We found that VitD treatment of psoriatic lesional skin upregulated BACH2 expression. Both haploinsufficiency and single nucleotide variants of BACH2 associate with monogenic and polygenic autoimmunity, respectively, in humans36,47. No BACH2 knockout humans have yet been identified, suggesting incompatibility of complete BACH2 deficiency with life. Indeed, Bach2–/– mice succumb to fatal autoimmunity. We found that loss of even 50% of the normal cellular concentration of BACH2 in the haploinsufficient state substantially altered (~70% of) the VitD-regulated transcriptome. As only a proportion of these genes were directly BACH2-bound, it is probable that BACH2 is a requisite for normal recruitment and function of the other transcriptional regulators.
Both the incidence and severity of COVID-19 are epidemiologically associated with VitD deficiency/insufficiency19, but the molecular mechanisms remain unknown. We found a link between the inflammatory TH1 program and a VitD-repressed gene set. Attempts to study CD4+ T cells from the site of inflammation were unsuccessful due to the rapid apoptosis of patient cells, but our in silico analyses suggest either dysregulation of the VitD program in COVID-19 or that simple deficiency/insufficiency of substrate (VitD) might explain the epidemiological association.
IL-6 is a pleiotropic, often pro-inflammatory, cytokine. IL-6 is implicated in the COVID-19 ‘cytokine storm’ and targeting of this cytokine specifically has proved beneficial to patients32. Our data suggest that pro-inflammatory IL-6 functions may be redirected to production of anti-inflammatory IL-10 by VitD in activated human helper T cells. VitD supplementation in children significantly increases serum IL-6 (and nonsignificantly increases IL-10)48 indicating that these observations may also occur in vivo. In the skin, where VitD concentrations are high, IL-6 overexpression protects from injurious stimuli or infection49 and IL-6-deficiency impairs wound healing50. Moreover, an adverse effect of anti-IL-6R for treating inflammatory arthritis is idiosyncratic development of psoriasis51, indicating a tolerogenic role for IL-6 at this site. Thus, adjunct VitD therapy in severe COVID-19 could potentially divert pro-inflammatory and induce anti-inflammatory effects of IL-6, which may be an alternative to blocking IL-6R signaling.
These data identified the VitD pathway as a potential mechanism to accelerate shutdown of TH1 cells in severe COVID-19. From experience in other diseases, it is likely that VitD will be ineffective as monotherapy. Combination therapy could potentially ameliorate significant adverse effects of other drugs, for example high-dose corticosteroids, including over-immunosuppression or metabolic side-effects. An important consideration of VitD therapy in COVID-19 is stimulation of IL-6 production from CD4+ T cells. Although autocrine/paracrine IL-6 induces IL-10 in these cells, IL-6 could potentially have pro-inflammatory properties on other cells. These possibilities may be mitigated by adding VitD as an adjunct to other immunomodulators, such as corticosteroids or JAK inhibitors13. Of note, two randomized clinical trials with calcifediol, a VitD analog with high bioavailability not requiring hepatic 25-hydroxylation, comprising >1,000 patients together, reported reductions in risk of intensive-care unit admission or death when used in addition to standard care (odds ratio of 0.13 and 0.22, respectively52,53). These findings are not necessarily specific to COVID-19, as VitD can protect against acute respiratory tract infections in general17.
In conclusion, we identified an autocrine/paracrine VitD loop permitting TH1 cells to both activate and respond to VitD as part of a shutdown program repressing IFN-γ and enhancing IL-10. These events involved significant epigenetic reshaping and recruitment of a network of key TFs. These pathways could potentially be exploited therapeutically to accelerate the shutdown program of hyper-inflammatory cells in patients with severe COVID-19.