Inherited salt-losing tubulopathies are associated with immunodeficiency due to impaired IL-17 responses

Increased extracellular sodium activates Th17 cells, which provide protection from bacterial and fungal infections. Whilst high salt diets have been shown to worsen autoimmune disease, the immunological consequences of clinical salt depletion are unknown. Here, we investigate immunity in patients with inherited salt-losing tubulopathies (SLT). Forty-seven genotyped SLT patients (with Bartter, Gitelman or EAST Syndromes) are recruited. Clinical features of dysregulated immunity are recorded with a standardised questionnaire and immunological investigations of IL-17 responsiveness undertaken. The effects of altering extracellular ionic concentrations on immune responses are then assessed. Patients are hypokalaemic and hypomagnesaemic, with reduced interstitial sodium stores determined by 23Na-magnetic resonance imaging. SLT patients report increased mucosal infections and allergic disease compared to age-matched controls. Aligned with their clinical phenotype, SLT patients have an increased ratio of Th2:Th17 cells. SLT Th17 and Tc17 polarisation is reduced in vitro, yet STAT1 and STAT3 phosphorylation and calcium flux following T cell activation are unaffected. In control cells, the addition of extracellular sodium (+40 mM), potassium (+2 mM), or magnesium (+1 mM) reduces Th2:Th17 ratio and augments Th17 polarisation. Our results thus show that the ionic environment typical in SLT impairs IL-17 immunity, but the intracellular pathways that mediate salt-driven Th17 polarisation are intact and in vitro IL-17 responses can be reinvigorated by increasing extracellular sodium concentration. Whether better correction of extracellular ions can rescue the immunophenotype in vivo in SLT patients remains unknown.

G lobally, dietary salt (sodium chloride) intake exceeds the amount that we consumed in our relatively recent evolutionary past, and in most communities is greater than the World Health Organisation recommended limit of 2 g sodium per day 1,2 . Sodium intake is linked to the development of hypertension and cardiovascular disease, but other adverse effects of excess sodium are beginning to emerge. These include an increasing awareness that sodium directly impacts immunity and is associated with the development of inflammatory and autoimmune disease 3,4 .
IL-17 is a pro-inflammatory cytokine produced by CD4 + (Th17) and CD8 + (Tc17) T cells. IL-17 responses provide protection from infection, particularly at epithelial surfaces, but are increasingly implicated in autoimmune disease 5 . Inherited defects in IL-17 immunity lead to chronic mucocutaneous candidiasis (CMC), which is characterised by mucosal bacterial and fungal infection, often associated with other clinical features of dysregulated immunity including increased allergic disease. CMC is most commonly due to inherited mutations affecting the phosphorylation and activity of the cytokine signalling molecules Signal transducer and activator of transcription 1 and 3 (STAT1 and STAT3), which subsequently impacts Th17 polarisation 6,7 .
Chronic salt loading leads to sodium storage in muscles and the skin, resulting in interstitial sodium concentrations at these sites in excess of those in plasma 8 . Sodium here may be cleared by macrophages and this finding provides the initial evidence directly linking extracellular sodium to immune cell function 9 . Sodium has since been shown to impact multiple components of both innate and adaptive immunity 4,10 . Increased sodium promotes activation of pro-inflammatory cell subtypes such as Th17 cells and M1 macrophages, while inhibiting regulatory cells [11][12][13][14] . Intracellular pathways that mediate this inflammatory effect of sodium are beginning to emerge. In Th17 cells, this involves upregulation of nuclear factor of activated T cells 5 (NFAT5) and serum/glucocorticoid-regulated kinase 1 (SGK1) 12,13 . The consequences of salt-driven activation of these inflammatory cells are increased autoimmunity, exacerbated transplant rejection, and increased clearance of cutaneous infection [11][12][13][15][16][17][18] .
Renal sodium handling involves the glomerular filtration of large amounts of sodium, which is then reabsorbed across the tubular epithelium, a process that is homeostatically controlled mainly in the distal nephron. This involves a number of different sodium transporters often functioning in concert with other ion transport mechanisms 19 . Inherited defects in renal sodium reabsorption due to mutations in genes encoding sodium or other ion transporters underlie the salt-losing tubulopathies (SLT) 20 . The commonest causes of SLT are defects in sodium reabsorption in the thick ascending limb (TAL) and distal convoluted tubule (DCT), leading to Bartter syndrome (BS) and Gitelman syndrome (GS), respectively. Defective reabsorption in the DCT is also a feature of the rarer EAST (Epilepsy, Ataxia, Sensorineural deafness, and Tubulopathy) Syndrome, which has a tubular phenotype indistinguishable from GS 21 . Together, these Salt-Losing Tubulopathies share a number of clinical and biochemical features including chronic salt depletion from renal salt wasting, in addition to hyperaldosteronism and hypokalaemic metabolic alkalosis, commonly associated with renal magnesium wasting and hypomagnesaemia. Patients often report a number of somewhat unexplained symptoms, including recurrent febrile episodes in childhood; a link to autoimmunity in both BS and GS has been described, but not fully explained [22][23][24][25][26][27][28] . Immune reactivity in SLT has not been previously explored.
To understand the consequences of chronic in vivo salt depletion, we investigate immunity in patients with SLT. We demonstrate that SLT is associated with clinical features of immunodeficiency due to reduced IL-17 responses. We provide data that suggest the immunophenotype in SLT is a consequence of chronic salt depletion and the associated changes in ionic environment in vivo, and highlight the important role of sodium in addition to other extracellular ions on immune cell activation states. In doing so, we describe a novel cause of IL-17 immunodeficiency and provide the basis for further study of altering extracellular ions to modify infection risk and as a potential immunosuppressive strategy in immune-mediated disease.
Total body salt depletion is suggested in SLT by low/normal blood pressure; mean systolic and diastolic blood pressures are 117 ± 12 and 74 ± 7.4 mmHg, respectively. Six SLT patients underwent 23 Na-MRI imaging of the lower limb, which confirms reduced sodium stores in the skin compared to age-matched healthy controls (HCs) (Fig. 1).
Reduced IL-17 responses underlie CMC, which is commonly due to mutations in STAT1 or STAT3 and may be part of the Hyper-IgE syndrome. To assess whether the infections suffered by SLT patients are consistent with an impairment in IL-17 immunity, we devise a scoring system based on the infectionrelated components of the Hyper-IgE syndrome diagnostic criteria, and apply this to SLT patients and controls (Supplemental Table 5) 31 . IL-17-related infection scores are higher in SLT patients than both control groups (Fig. 2a); moreover, the clinical phenotype of SLT patients is similar to patients with STAT1 and STAT3-mediated IL-17 defects (Supplemental Table 6).
SLT patients have an altered urinary microbial community. Given the increased prevalence of SLT mucosal microbial infections, we were interested to understand if there were differences in the microbial community in SLT patients at one of the common sites of infection. Hence, we perform urine sediment cultures, which demonstrate there are altered bacterial constituents with an increase in the presence of Corynebacterium sp. in SLT patients compared to controls (Supplemental Tables 7  and 8). To determine whether alterations in extracellular sodium concentration directly affect microbial growth, strains of C. amycolatum as well as other commonly encountered microbes (E. coli, S. aureus, and C. albicans) are cultured in media supplemented with NaCl. NaCl has no effect on the growth of the bacteria tested, whereas there is a dose-dependent reduction in the size of Candida colonies with additional NaCl (Supplemental Fig. 2). A direct effect of altered extracellular sodium may, therefore, contribute to mucosal fungal infection; however, the lack of an effect of an altered extracellular environment on bacterial growth supports a primary defect in immunity underlying the altered microbiome and increased urinary infection rate observed in SLT.
Initial immunological analysis of SLT patients. Immunological analysis of SLT patients is summarised in Supplemental Table 9. Total IgE level is elevated in 10 (33.3%) of 30 patients in whom it was measured, and CD16 + CD56 + NK (natural killer) cells are reduced in 13 (40.6%) of 32 patients (Fig. 2b, c). Otherwise, parameters tested are within the laboratory reference range.
T-cell proliferation and activation, determined as the percentage of CD4 + cells expressing Ki67 and CD25, respectively, after 72 h of anti-CD3 and anti-CD28 stimulation, are no different in SLT patients compared to controls (Fig. 2d, e). Analysis of antigenspecific antibody responses demonstrates SLT patients have protective levels of varicella zoster and pneumococcal immunoglobulin G, similar to the healthy adult population (Fig. 2f). Moreover, CD4, CD8, CD45RA, and CD45RO subsets are not different in SLT patients compared to controls (Supplemental Fig. 3). Initial immunological analysis of SLT patients, therefore, demonstrates no gross defects in adaptive immunity.
SLT patients have reduced IL-17 responses. Given the clinical findings in SLT of increased mucosal infection, and the known effect of salt on Th17 responses, we assessed CD4 + T cell subsets in SLT and compare these to healthy and disease controls. Analysis of IFNγ, IL-4, and IL-17 expression in CD4 + cells, representative of Th1, Th2, and Th17 cells, respectively, demonstrates a significant reduction of Th17 cells in SLT, whereas Th1 and Th2 cells are not different between groups (Fig. 3a, b). When CD4 subsets are expressed as a ratio of one cell subtype to another within each subject, there is an increase in the ratio of Th2:Th17 cells in SLT  observed findings of increased infection and allergy rate in the SLT cohort (Fig. 3c).
To further investigate IL-17 responses, peripheral blood mononuclear cells (PBMCs) are cultured under optimal Th17 polarising conditions for 7 days. IL-17 expression in CD4 + and CD4 − cells, representing Th17 and Tc17 polarisation, respectively, is reduced in SLT patients compared to both healthy and disease controls (Fig. 4a, b, d). Supernatant IL-17 concentrations are reduced in SLT patients compared to HCs (Fig. 4c). Together with the CD4 + cell subset analysis, these data confirm SLT patients have reduced IL-17 responses.
Altered STAT1 and STAT3 phosphorylation are a common cause of inherited defects in IL-17 immunity. We assess STAT1 and STAT3 phosphorylation in SLT lymphocytes, but these are normal demonstrating STAT1 and STAT3 signalling defects are not the cause of defective IL-17 responses in SLT patients (Fig. 5a Mutations in a number of different ion channels expressed on immune cells lead to immunodeficiency, largely as a result of associated changes in calcium signalling during leucocyte activation 32 . The commonest defective sodium transporter in our SLT cohort was the sodium chloride cotransporter (NCC, SLC12A3). We investigate NCC expression on immune cells and assess whether its inhibition leads to reduced IL-17 responses. NCC is expressed on diverse immune cell subtypes but NCC inhibition with hydrochlorothiazide (HCT; 20 μM) has no effect on T-cell calcium flux or in vitro IL-17 responses in cells from HCs ( Fig. 5c and Supplemental Fig. 4D-F). SLT patients have a normal pattern of calcium flux after T-cell activation (Fig. 5d, e). Furthermore, infection score and IL-17 responses are not different according to the ion transporter affected (Supplemental Fig. 4G). Given these data, reduced IL-17 responses in SLT are not likely to be due to primary perturbations of NCC function or secondary dysregulation of intracellular calcium signalling in immune cells.  sodium, potassium, and magnesium. We therefore assess the effect of altering extracellular sodium, potassium, and magnesium concentrations, individually and in combination, on the balance of CD4 + subsets after non-specific T-cell stimulation and on IL-17 responses in control cells cultured under optimal polarising conditions.
We first measure electrolyte concentrations of XVIVO15 media to determine baseline conditions ( Supplementary Fig. 5A). We then stimulate PBMCs with anti-CD3 and anti-CD28 for 72 h in different ionic environments and measure the expression of IFNγ, IL-4, and IL-17 in CD4 + cells. The addition of NaCl (0-40 mM), KCl (0-2 mM), and MgCl 2 (0-1 mM) results in a dosedependent reduction in the ratio of expression of IFNγ:IL-17 and IL-4:IL-17 in CD4 + cells (Fig. 6a-c). The addition of each of the ions has no effect on cell viability ( Supplementary Fig. 5B), there is no consistent effect on IFNγ:IL-4 expression, and combining multiple ions exacerbates IL-4:IL-17 expression ratio (Fig. 6d). Hence, SLT patients have an extracellular ionic environment, which favours Th2 over Th17 responses, as we had found in our CD4 subset analysis and consistent with their clinical phenotype.
We then assess the effect of altering extracellular ionic concentrations on 7-day IL-17 responses. We demonstrate a dose-dependent increase in Th17 and Tc17 polarisation with the addition of NaCl (0-40 mM), KCl (0-2 mM), and MgCl 2 (0-0.8 mM) (Fig. 7a-c). Addition of ions has no effect on cell viability or IFNγ responses (Supplemental Fig. 6A, B). We interrogate the NaCl responses more closely and demonstrate that the polarising effect of NaCl on Th17 and Tc17 cells is consistent in HCs (Fig. 7d, e), that its effect is predominantly mediated by the sodium concentration rather than osmolality (Supplemental Fig. 6C), and that sodium affects both naive and memory T-cell populations (Supplemental Fig. 6D-F). Angiotensin II added to culture conditions increases IL-17 responses whereas aldosterone has no effect (Supplemental Fig. 6G). SLT patients, therefore, have an extracellular ionic environment that reduces IL-17 responses, and this environment may impact IL-17 expression in multiple cell types. Angiotensin II may serve as a compensatory immune mechanism for salt loss.
We then correlate serum biochemical and clinical parameters in SLT with their IL-17-related infection score. Impaired renal function as demonstrated by increased serum creatinine leads to reduced salt loss and blood pressure may act as a surrogate for sodium stores 33,34 . In support of a predominant effect of salt depletion and reduced extracellular sodium on immunity, blood pressure and serum creatinine inversely correlate most closely with infection score (diastolic blood pressure Pearson r −0.37 (95% CI −0.60, −0.09), p = 0.013; mean arterial pressure Pearson whereas there is no correlation of infection score with serum magnesium or potassium (Supplemental Fig. 7A, B). Moreover, aligned with their differences in renal function, there is a difference in infection score between SLT types grouped according to biochemical phenotype, with infection score highest in patients with GS and EAST syndrome (distal tubule dysfunction and most preserved renal function), and lowest in patients with BS types 1, 2, and 4 (loop dysfunction and most impaired renal function) ( Fig. 7f, g).
SLT IL-17 responses can be rescued in vitro by salt. The intracellular pathway by which sodium increases Th17 polarisation includes the upregulation of SGK1, NFAT5, and the IL23receptor (IL-23R) 12,13 . We assess whether this pathway is intact in SLT patients. We demonstrate that SLT lymphocyte expression of SGK1 and NFAT5 are not reduced compared to HCs, neither is IL-23R expression on CD4 + cells ( Fig. 8a-e); IL23-R expression is in fact increased in non-CD4 lymphocytes in SLT patients ( Fig. 8B and Supplemental Fig. 8A).
Given the preservation of this pathway, we hypothesised that in vitro IL-17 responses can be rescued in SLT patients by supplementing culture conditions with salt. Indeed, the addition of 40 mM NaCl to culture conditions upregulates IL-17 responses in SLT such that Th17 and Tc17 polarisation are equivalent to healthy and disease control polarisation under standard conditions ( Fig. 8f, g). Salt responsiveness of in vitro IL-17 responses in SLT patients is no different to controls, and there is no difference in salt responsiveness according to SLT type (Supplemental Fig. 8B-D). These data demonstrate that salt supplementation ameliorates IL-17 response defects in SLT in vitro, but whether this is feasible or effective clinically in vivo is unknown.
Innate immune responses in SLT patients. Polarisation of Th17 cells requires antigen presentation by antigen-presenting cells in the presence of Th17 polarising cytokines produced by innate immune cells. Defective IL-17 responses can, therefore, be a consequence of impaired innate immunity. We assess innate immune responses in SLT patients by measuring cytokine production in response to a range of stimuli in whole blood assays. We demonstrate no reduction in innate responses; indeed, production of several innate cytokines (e.g. IL-1, IL-6, and TNF) is increased in SLT patients compared to controls ( Table 2).
We subsequently analyse monocyte responses in more detail in SLT patients. We find no difference in monocyte subsets (classical, intermediate, and non-classical) or in monocyte expression of TNF in SLT patients compared to controls in the experiment we use (Supplemental Fig. 9A-C). Moreover, adding 40 mM NaCl, 2 mM KCl, 1 mM MgCl 2 , or aldosterone NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18184-3 ARTICLE (10-100 nM) to stimulating conditions has no effect on monocyte TNF expression (Supplemental Fig. 9D). Reduced innate immune responses are therefore not the cause of reduced IL-17 responses and increased infection in SLT patients. We finally conduct an additional analysis of NK cells in SLT to further characterise NK cell subsets and assess NK cell function. In this analysis, CD45 + CD3 − CD56 + CD16 + (NK) cells account for 10.2% (5.9-13.8) of lymphocytes in HCs and 6.9% (3.9-10.0) in SLT patients (p = 0.1, Supplemental Fig. 10A). 8 (50%) SLT patients have NK cells of less than 7% of lymphocytes, but so do 5 (29%) controls (p = 0.3). The proportion of CD56 + cells, however, expressing CD16 (the Fc receptor) is reduced in SLT patients (Supplemental Fig. 10B, D). To assess NK cell function, PBMCs are stimulated with IL-12 and IL-18 for 4 h and NK cell expression of IFNγ determined. IFNγ expression is not different between SLT and controls (Supplemental Fig. 10C).
Although the effect of extracellular sodium has been assessed in large parts of the immune system, its effect on NK cells has not. We, therefore, stimulate NK cells in the presence of additional NaCl (+40 mM). Adding NaCl to stimulating conditions increases the proportion of lymphocytes expressing CD56, but IFNγ expression by CD56 + cells is reduced (Supplemental Fig. 10E, F). There is therefore a trend to a reduction in NK cell number in SLT patients with reduced expression of the Fc receptor on CD56 + cells, demonstrating NK cells in SLT patients have a tolerant phenotype.

Discussion
Studies to date on the effect of altered sodium balance on immunity have largely focused on salt loading and how this impacts inflammatory or autoimmune disease. Alongside the inflammatory effect of sodium demonstrated on multiple immune cells in vitro and in animal models, increased sodium intake in patients has been linked to the development of rheumatoid arthritis, correlates with disease severity in multiple sclerosis, and worsens renal allograft outcomes independently of any blood pressure effect [35][36][37] . These data imply that reducing salt intake also impacts immunity and may ameliorate diverse inflammatory states, but this hypothesis is untested and the immunological consequences of chronic salt depletion are unknown. Moreover, the evolutionary pressures linking immune function to sodium are unclear. Long-term salt restriction studies are logistically challenging and difficult to adhere to; therefore to understand the consequences of reduced sodium balance, we investigated immunity in patients with rare inherited SLTs. These patients provide a unique in vivo model of chronic salt depletion and an assessment of their immunity was previously unexplored.
Novel findings from long-term sodium balance studies have demonstrated a third compartment of sodium stored at interstitial sites in the muscle and skin. It is proposed that the skin contains an osmotic gradient-generating system akin to the countercurrent mechanism in the kidney 38  may also provide protection from infection 11,18 . Although the sodium transport mechanisms in the kidney are well described, ion channels and transporters involved in skin sodium transport are largely unknown. Visualisation and measurement of skin sodium stores with magnetic resonance imaging ( 23 Na-MRI) has demonstrated that sodium concentrations at these sites increase with age and in disease states that result in sodium loading such as heart failure and chronic kidney disease 33  specific (BS1) and systemic (GS) transporters suggesting the predominant reason for reduced skin sodium is renal sodium wasting as opposed to a primary defect in sodium transport in the skin. This alteration in interstitial sodium combined with reduced serum potassium and magnesium concentrations mean immune cells in SLT patients are exposed to a unique combination of altered extracellular ionic cues, which we propose impacts immune function.
The presentation of SLT patients may be through the identification of biochemical abnormalities incidentally on blood tests performed for other reasons, or with an array of relatively nonspecific symptoms. These include cramps, weakness, dizziness, and fatigue, and such symptoms have been traditionally attributed directly to electrolyte derangement or relative hypovolaemia. However, there are no consistent data demonstrating a correlation between symptom burden and electrolyte abnormalities, and symptoms are often persistent and disabling even when electrolytes are replete. Moreover, some recognised clinical features, such as febrile episodes in childhood in GS, theoretically cannot be explained by a direct electrolyte effect 27,[43][44][45] . Our data demonstrating immunodeficiency in SLT provide an explanation for these symptoms, and we describe novel immune-mediated features that have not been appreciated in SLT patients before. In our cohort, the persistent nature of bacterial and fungal infections alongside recurrent allergic episodes carried significant morbidity. Moreover, an association between SLT and autoimmunity (e.g. autoimmune thyroid disease, Sjögren's syndrome, autoimmune hepatitis, and vasculitis) is recognised [22][23][24]26,28 ; findings in our cohort confirm this association and our demonstration of dysregulated immunity provide an explanation for this link.
Th17 cells are pro-inflammatory, and provide protection against extracellular bacterial and fungal infections, particularly at mucosal surfaces 46 . Polarisation requires stimulation by IL-1β, IL-6, and TGF-β, whereas full maturation requires IL-21 and IL-23. Th17 cells secrete various cytokines including IL-17, which induces release of other pro-inflammatory cytokines and chemokines, and is important for neutrophil recruitment. Defective IL-17 responses may be inherited and result in CMC syndrome, or may be acquired in patients with thymoma 47 (production of neutralising antibodies to IL-17) or in patients taking anti-IL-17 monoclonal antibodies for the treatment of autoimmune disease 48 . We demonstrate that the primary cause for immunodeficiency in SLT patients is reduced IL-17 responses and the mucosal nature of bacterial and fungal infections in SLT is consistent with the phenotype of patients with inherited IL-17 defects (Supplemental Table 6). Syndromic CMC is most commonly due to STAT1 phosphorylation abnormalities 6 , but these were absent in SLT patients, as were alterations in STAT3mediated cytokine signalling 31,49 . We describe, therefore, a novel cause of IL-17 immunodeficiency, through an ionic mechanism not previously reported before.
Immunodeficiency as a result of altered ionic flux in immune cells is largely described in humans from inherited ion channelopathies that result in impaired store-operated calcium entry (SOCE) during T-cell activation 50 . This may be due to calcium release-activated calcium current (CRAC) channel mutations leading to impaired calcium release from the endoplasmic reticulum (e.g. stromal interaction molecule 1 [STIM1] mutations) or impaired extracellular calcium entry across the plasma membrane (e.g. calcium release-activated calcium modulator 1 [ORAI1] mutations) [51][52][53][54][55][56] . Impaired SOCE also can result from a magnesium channel defect due to pathogenic mutations in Magnesium Transporter 1 (MAGT1), which results in X-linked immunodeficiency with magnesium defect, EBV viraemia and neoplasia (XMEN) syndrome 57,58 . Moreover, in mice, impaired SOCE due to deletion of ORAI1 or STIM1 impairs Th17 polarisation 59,60 , and sodium has been shown to exert its pro-inflammatory effect in dendritic cells through epithelial sodium channel (ENaC) flux altering calcium signalling 61 . In SLT, we demonstrate that calcium flux is unaffected after T-cell activation and patients with inactivating ENaC mutations (pseudohypoaldosteronism type 1) were not included in the SLT cohort. Although we have demonstrated the presence of other sodium transport mechanisms on diverse immune cells, this unaltered calcium flux, alongside the lack of an effect of sodium chloride cotransporter inhibition on Th17 polarisation, and the inclusion of patients with multiple ion channel/transporter mutations make reduced function of channel/transporter activity on immune cells an unlikely reason for altered immunity.
SLT patients are unique in their characteristic extracellular ionic environment, directly and indirectly related to renal salt wasting. In the light of recent evidence highlighting the effect of sodium on IL-17 responses 12,13,16,17,62,63 , and on previous evidence demonstrating an effect of other extracellular ions (e.g. magnesium) on immunity 64 , we hypothesised that reduced IL-17 responses are due to the altered ionic environment in vivo in SLT patients. We demonstrated that the addition of sodium, potassium and magnesium, within a clinically relevant range, promoted both a reduction in the ratio of Th2:Th17 polarisation during 3-day T-cell stimulation experiments, and an increase in Th17 and Tc17 polarisation during 7-day experiments in culture conditions optimal for IL-17 production. These data show that the extracellular ionic environment typically found in SLT patients is inhibitory to protective IL-17 responses. Our experiments confirm previous data demonstrating sodium promotes IL-17 responses 12,13,16,17,62,63 , but also highlight a potential role of sodium impacting Th2 and NK responses, and suggest an influence of other cations on Th17 polarisation. We also provide evidence for the first time that sodium depletion affects immunity and leads to IL-17-mediated immunodeficiency. Moreover, within the SLT cohort, blood pressure and serum creatinine Fig. 5 Initial investigation of impaired IL-17 responses in salt-losing tubulopathy patients. a Phosphorylation of STAT3 in CD4 + cells after stimulation with IL-6 and IL-21 in SLT patients (n = 13). Phosphorylation is expressed as ratio of upregulation of pSTAT3 in SLT compared to the HC assessed in the same experiment (n = 4 HCs assessed across 4 experiments). Red line drawn at ratio of 1 representing no difference between SLT and HC. b Phosphorylation of STAT1 in CD4 + cells after stimulation with IFNα in SLT patients (n = 13). Phosphorylation is expressed as ratio of upregulation of pSTAT1 in SLT compared to the HC assessed in the same experiment (n = 4 HCs assessed across 4 experiments). Red line drawn at a ratio of 1 representing no difference between SLT and HC. c Th17 and Tc17 polarisation, and supernatant IL-17 concentration in cell culture experiments (11 experiments in 11 healthy controls) with the addition of hydrochlorothiazide (HCT) 20 μM to culture conditions. Each variable is expressed as a ratio to the readout in standard culture conditions; red line drawn at ratio of 1 representing no difference in readouts with the addition of HCT. d Area under the calcium flux curve in healthy controls (n = 13) and Gitelman syndrome patients (n = 5) (as determined using the ratiometric calcium dye Indo-1 gated on CD4 + cells) after T-cell activation with anti-CD3: HC 275.9 AU (265.9-279.9); and Gitelman Syndrome (GS) 277.8 AU (257.5-280.5), p = 0.94. Compared with a two-sided Mann-Whitney test. Error bars represent interquartile range around the median. e Representative calcium flux curves in CD4 + cells after T-cell activation in a healthy control and two patients with GS. ns not significant (p > 0.05), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, SLT salt-losing tubulopathy, HC healthy control, DC disease control, GS Gitelman syndrome. Source data are provided as a Source data file.   inversely correlated to infection score, and hence we propose that extracellular sodium as opposed to alterations in other ions is the main determinant of infection risk. This requires confirmation with direct measurement of sodium stores in a larger number of SLT patients as part of future study. Given this proposal of ionic changes altering immunity in SLT, we lastly tested whether manipulation of the extracellular ionic environment in vitro could be used to augment SLT IL-17 responses, as we had shown in controls. We demonstrated that the intracellular pathway that mediates sodium-driven Th17 polarisation is intact in SLT patients and that Th17 polarisation could be rescued with the addition of NaCl to culture conditions. This raises the possibility that better correction of extracellular ionic concentrations in vivo in SLT patients may improve IL-17 responses and mitigate infection risk and warrants further study. Moreover, our data provide the basis for investigating whether manipulation of the extracellular ionic environment may be used as a therapeutic strategy in IL-17-mediated inflammatory diseases.
We demonstrated that SLT patients do not have alterations in naive and memory cell populations, and that the extracellular ionic environment impacts memory as well as naive cells confirming recent reports 65 . Hence, we used PBMCs as opposed to isolated cells for IL-17 polarisation experiments. We did not assess all CD4 + subtypes and any effect on regulatory T cells or T follicular helper cells, both of which may be affected by sodium 14,15,66 , in SLT patients was unexplored. Similarly, sodium has been shown to impact dendritic cell function 61 , but the clinical phenotype of SLT patients (absent recurrent viral and mycobacterial infections) and the specific defect in IL-17 responses were inconsistent with primary defects in dendritic  67,68 . Moreover, innate cytokine responses in whole blood assays were unaffected and hence further functional assessment of dendritic cells was not undertaken. We measured calcium flux during T-cell activation but were unable to measure sodium flux in patients or controls and hence were unable to confirm that SLT patients do not have a primary defect in ion transport mechanisms on immune cells. SLT patients have an activated renin-angiotensin-aldosterone system, albeit largely as a consequence of salt wasting, and while we assessed the effect of aldosterone and angiotensin II on Th17 polarisation, we did not assess the direct effect of renin, which has been shown to have an independent immunomodulatory role 69 . We also did not assess the impact of altered prostaglandin E2, which is known to affect immunity 70 and which is increased in some patients with BS types 1, 2, and 4. Finally, we did not investigate the in vivo location of the effect of sodium and other ions on immunity, which remains an important unknown from this and other studies on the effect of salt on immunity.
In conclusion, we describe a novel immunodeficiency in SLT patients, who have increased mucosal bacterial and fungal infections, alongside other clinical features of dysregulated immunity including autoimmune phenomena, dermatitis, and allergic disease. These clinical features are associated with impaired IL-17 responses, which we propose are due to sodium wasting and the unique extracellular ionic environment found in SLT patients. We highlight the role of multiple extracellular ions on immune cell activation states and demonstrate that SLT IL-17 responses may be rescued in vitro by salt. The manipulation of the extracellular ionic environment may, therefore, be of therapeutic benefit in SLT patients to mitigate infection risk.

Methods
SLT and control cohorts. Patients with genotyped BS, GS, or EAST syndrome were recruited from the Royal Free Hospital and Great Ormond Street Hospital Tubular Disorders Clinics, London, UK. Demographic, biochemical, and clinical data on the day of recruitment were recorded. Biochemical parameters were compared between patients with loop dysfunction (BS types 1, 2, and 4), distal tubule dysfunction (GS and EAST syndrome), and a mixed phenotype (BS type 3).
SLT patients underwent a structured clinical history focused on clinical features of dysregulated immunity. This was also undertaken in age and sex-matched healthy (n = 24) and disease (n = 22) controls. Disease controls included patients attending the Tubular Disorders Clinics, but who had no evidence of a significant salt-wasting phenotype. These patients were recruited from the same clinics as the SLT patients, and they underwent the same structured clinical history and biochemical analysis. Conditions included: proximal Tubulopathy = 7; monogenic hypertension = 5 (familial hyperkalaemic hypertension = 3; STX16 mutation = 2); distal renal tubular acidosis/nephrocalcinosis = 3; isolated renal magnesium wasting = 1; hyperparathyroidism = 1; tubulointerstitial nephritis = 2; medullary sponge kidney = 1; diabetes insipidus = 1; non renal hypokalaemia = 1 (Supplementary Table 2). Healthy controls were volunteers who worked within the hospital or university and who had no known medical conditions. The prevalence of each clinical variable was compared between SLT and control groups.
An infection score was subsequently determined for each subject related to infections known to arise from defects in IL-17-mediated immunity. To do this we used the infection-related scoring system in the diagnostic criteria of Hyper-IgE syndrome, which results from inherited defects in IL-17 immunity (Supplemental Table 5). IL-17-related infection scores were compared between SLT and controls. We also compared infection scores between SLT subtypes (according to defective transporter and biochemical group) and we correlated infection score to serum biochemical and clinical parameters in SLT patients. 23 Na-MRI imaging. To determine skin and muscle sodium storage, SLT patients (n = 6) and HCs (n = 4) underwent 23 Na-MRI imaging of the lower right limb. Tubes of known 23 Na concentration (0, 20, 40, 60, and 80 mM, respectively) were placed on the participants' limb within the field of view and were used for quantification. All participants were scanned on a 3 T mMR Biograph with a transmit/ receive birdcage 23 Na coil (Stark Contrast MRI Coils Research). Three sets of 23 Na weighted MR images were acquired with a balance steady-state free precision (BSSFP) sequence (Bandwidth = 289 Hz/pixel, Echo Time = 3.33 ms, Repetition Time = 21 ms, Signal Averages = 100, resolution = 3.1 × 3.1 × 20mm 3 , acquisition time = 2:17). Quantified 23 Na maps were calculated with an in-house script (Matlab). Regions of Interests were drawn per acquisition per participant on each sodium phantom and the averaged signal per phantom was used to calculate a calibration curve as: [Known 23 Na Concentration] = p1*[Averaged signal]+p2, where p1 and p2 are the slope and intercept, respectively. The values of p1 and p2 were then used to calculate the 23 Na concentration per pixel using: [ 23 Na Concentration in mM] = ([Signal] -p2)/p1.
Mean sodium concentrations in the skin and in 4 muscle groups were determined for each patient. Image analysis was performed using ITK-Snap v3.0 software (http://www.itksnap.org). Mean sodium concentrations in the skin and muscles were compared between SLT patients and controls.
Urine culture experiments. To characterise the urinary microbial community, urine specimens were collected from SLT patients (n = 14) and HCs (n = 12), and sediment cultures undertaken. A 20 ml volume urine specimen was collected and cells pelleted by centrifugation. Cell pellets were resuspended in 400 µl of phosphate buffered saline (PBS) (Life Technologies, UK) and ten-fold serial dilutions were performed in PBS to facilitate the quantification of microbial colonies. Cultures were kept in an ordinary incubator at 37°C for 18-24 h. Following incubation, the resulting microbial growth was quantified in colony-forming units per millilitre (cfu/ml).
Cultured organisms were grown separately on Columbia blood agar (Oxoid, UK) for species-level identification using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS). The direct colony protocol was adopted on the MicroFlex LT mass spectrometer (Bruker Daltonics, USA). Briefly, each cultured isolate was spotted on a 96-well target plate and overlaid with 1 µl matrix solution consisting of alpha-cyano-4-hydroxycinnamic acid dissolved in 50% acetonitrile and 2.5% trifluoroacetic acid. Once air-dried for 5 min, the target plate was loaded and analysed using the MALDI Biotyper 3.0 software programme containing the Bruker Taxonomy 16S reference library. The number of different isolates identified, the microbial burden of each isolate, Groups are compared using a Kruskal-Wallis test with Dunn's multiple comparison testing (shown with significance bars). Error bars represent interquartile range around the median. g Tc17 polarisation in HC (n = 27) and SLT patients (n = 25) in standard media, and in SLT patients in media supplemented with 40 mM NaCl: HC + 0 mM NaCl 1.5% (0.6-3.0); SLT + 0 mM NaCl 0.6% (0.3-1.1); SLT + 40 mM NaCl 1.6% (0.9-3.1); p = 0.0007; groups are compared using a Kruskal-Wallis test with Dunn's multiple comparison testing (shown with significance bars). Error bars represent interquartile range around the median. ns not significant (p > 0.05), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. HC healthy control, SLT salt-losing tubulopathy, AU arbitrary units. Source data are provided as a Source data file. and the microbial identity of the isolates was recorded for each patient and summarised and compared between cohorts.
To determine if increasing extracellular sodium directly affected microbial growth, strains of Escherichia coli, Staphylococcus aureus, Candida albicans, and Corynebacterium amycolatum were grown on chromogenic agar (Biomerieux, France) in an ordinary incubator at 37°C. A bacterial suspension in sterile distilled water with turbidity equivalent to a 0.5 McFarland standard was made for each strain. Seven ten-fold serial dilutions were made for each strain and 25 µl of each dilution, along with the undiluted bacterial suspension, were plated onto one quadrant of four tryptone soya agar plates, which had been prepared with and without additional sodium chloride (unadjusted plate, +40 mM, +80 mM, +160 mM). Plates were incubated at 37°C and microbial growth was examined by visual inspection after 24 h.
Initial immunological analysis. Initial immunological analysis of SLT patients was undertaken in the National Health Service clinical laboratories of the Royal Free Hospital, London. This included full blood count, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), immunoglobulin A, G, M, and E concentrations, complement protein C3 and C4 concentrations, lymphocyte counts and subsets (CD3 + , CD4 + , CD8 + , CD19 + , CD16/56 + ).
Antibodies. Antibodies used are outlined in Supplementary Table 10.
Statistics. Data are presented as number and percentages for categorical variables, and mean and standard deviation or median and interquartile range for numerical variables depending on the distribution. Categorical variables were compared using the Fisher's exact or Chi-squared test. Non-parametrically distributed numerical variables were compared between two groups using the Mann-Whitney test and the Wilcoxon test for unpaired and paired data, respectively. Parametrically distributed numerical variables were compared with an unpaired or paired t-test. Variables are compared across greater than two groups with a one-way analysis of variance with multiple comparison testing; specifically, for non-parametric data, we used the Kruskal-Wallis test with Dunn's test for multiple comparisons. Numerical variables were correlated with a Pearson correlation. Unless otherwise specified, graphs plot individual points alongside median and interquartile range.
Analysis was performed using Graphpad Prism version 7 (www.graphpad.com). A p-value of ≤0.05 was considered statistically significant.
Study approval. The Royal Free Hospital Research and Development committee approved the study (identification number 7727) and all participants provided written informed consent prior to enrolment. Consent was also obtained to publish patient identifiable images where appropriate.