Bruton's tyrosine kinase inhibition attenuates disease progression by reducing renal immune cell invasion in mice with hemolytic-uremic syndrome.

Hemolytic-uremic syndrome (HUS) can occur as a complication of an infection with Shiga-toxin (Stx)-producing Escherichia coli. Patients typically present with acute kidney injury, microangiopathic hemolytic anemia and thrombocytopenia. There is evidence that Stx-induced renal damage propagates a pro-inflammatory response. To date, therapy is limited to organ-supportive strategies. Bruton's tyrosine kinase (BTK) plays a pivotal role in recruitment and function of immune cells and its inhibition was recently shown to improve renal function in experimental sepsis and lupus nephritis. We hypothesized that attenuating the evoked immune response by BTK-inhibitors (BTKi) ameliorates outcome in HUS. We investigated the effect of daily oral administration of the BTKi ibrutinib (30 mg/kg) and acalabrutinib (3 mg/kg) in mice with Stx-induced HUS at day 7. After BTKi administration, we observed attenuated disease progression in mice with HUS. These findings were associated with less BTK and downstream phospholipase-C-gamma-2 activation in the spleen and, subsequently, a reduced renal invasion of BTK-positive cells including neutrophils. Only ibrutinib treatment diminished renal invasion of macrophages, improved acute kidney injury and dysfunction (plasma levels of NGAL and urea) and reduced hemolysis (plasma levels of bilirubin and LDH activity). In conclusion, we report here for the first time that BTK inhibition attenuates the course of disease in murine HUS. We suggest that the observed reduction of renal immune cell invasion contributes - at least in part - to this effect. Further translational studies are needed to evaluate BTK as a potential target for HUS therapy to overcome currently limited treatment options.

Divergent roles of haptoglobin and hemopexin deficiency for disease progression of Shiga-toxin-induced hemolytic-uremic syndrome in mice.

Thrombotic microangiopathy, hemolysis and acute kidney injury are typical clinical characteristics of hemolytic-uremic syndrome (HUS), which is predominantly caused by Shiga-toxin-producing Escherichia coli. Free heme aggravates organ damage in life-threatening infections, even with a low degree of systemic hemolysis. Therefore, we hypothesized that the presence of the hemoglobin- and the heme-scavenging proteins, haptoglobin and hemopexin, respectively impacts outcome and kidney pathology in HUS. Here, we investigated the effect of haptoglobin and hemopexin deficiency (haptoglobin-/-, hemopexin-/-) and haptoglobin treatment in a murine model of HUS-like disease. Seven-day survival was decreased in haptoglobin-/- (25%) compared to wild type mice (71.4%), whereas all hemopexin-/- mice survived. Shiga-toxin-challenged hemopexin-/- mice showed decreased kidney inflammation and attenuated thrombotic microangiopathy, indicated by reduced neutrophil recruitment and platelet deposition. These observations were associated with supranormal haptoglobin plasma levels in hemopexin-/- mice. Low dose haptoglobin administration to Shiga-toxin-challenged wild type mice attenuated kidney platelet deposition and neutrophil recruitment, suggesting that haptoglobin at least partially contributes to the beneficial effects. Surrogate parameters of hemolysis were elevated in Shiga-toxin-challenged wild type and haptoglobin-/- mice, while signs for hepatic hemoglobin degradation like heme oxygenase-1, ferritin and CD163 expression were only increased in Shiga-toxin-challenged wild type mice. In line with this observation, haptoglobin-/- mice displayed tubular iron deposition as an indicator for kidney hemoglobin degradation. Thus, haptoglobin and hemopexin deficiency plays divergent roles in Shiga-toxin-mediated HUS, suggesting haptoglobin is involved and hemopexin is redundant for the resolution of HUS pathology.

A Synthetic Peptide Designed to Neutralize Lipopolysaccharides Attenuates Metaflammation and Diet-Induced Metabolic Derangements in Mice.

Metabolic endotoxemia has been suggested to play a role in the pathophysiology of metaflammation, insulin-resistance and ultimately type-2 diabetes mellitus (T2DM). The role of endogenous antimicrobial peptides (AMPs), such as the cathelicidin LL-37, in T2DM is unknown. We report here for the first time that patients with T2DM compared to healthy volunteers have elevated plasma levels of LL-37. In a reverse-translational approach, we have investigated the effects of the AMP, peptide 19-2.5, in a murine model of high-fat diet (HFD)-induced insulin-resistance, steatohepatitis and T2DM. HFD-fed mice for 12 weeks caused obesity, an impairment in glycemic regulations, hypercholesterolemia, microalbuminuria and steatohepatitis, all of which were attenuated by Peptide 19-2.5. The liver steatosis caused by feeding mice a HFD resulted in the activation of nuclear factor kappa light chain enhancer of activated B cells (NF-ĸB) (phosphorylation of inhibitor of kappa beta kinase (IKK)α/ß, IκBα, translocation of p65 to the nucleus), expression of NF-ĸB-dependent protein inducible nitric oxide synthase (iNOS) and activation of the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome, all of which were reduced by Peptide 19-2.5. Feeding mice, a HFD also resulted in an enhanced expression of the lipid scavenger receptor cluster of differentiation 36 (CD36) secondary to activation of extracellular signal-regulated kinases (ERK)1/2, both of which were abolished by Peptide 19-2.5. Taken together, these results demonstrate that the AMP, Peptide 19-2.5 reduces insulin-resistance, steatohepatitis and proteinuria. These effects are, at least in part, due to prevention of the expression of CD36 and may provide further evidence for a role of metabolic endotoxemia in the pathogenesis of metaflammation and ultimately T2DM. The observed increase in the levels of the endogenous AMP LL-37 in patients with T2DM may serve to limit the severity of the disease.

Involvement of NF-κB1 and the Non-canonical NF-κB Signaling Pathway in the Pathogenesis of Acute Kidney Injury in Shiga-toxin-2-induced Hemolytic-uremic Syndrome in Mice.

ABSTRACT: The hemolytic-uremic syndrome (HUS) is a thrombotic microangiopathy which can occur as a severe systemic complication after an infection with Shiga-toxin-(Stx)-producing Escherichia coli (STEC). Elevated levels of proinflammatory cytokines associated with the classical nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB) signaling pathway were detected in the urine of HUS patients. Thus, we hypothesize that the immune response of the infected organism triggered by Stx can affect the kidneys and contributes to acute kidney injury. Hitherto, the role of the classical and non-canonical NF-κB signaling pathway in HUS has not been evaluated systematically in vivo. We aimed to investigate in a murine model of Shiga toxin-induced HUS-like disease, whether one or both pathways are involved in the renal pathology in HUS. In kidneys of mice subjected to Stx or sham-treated mice, protein or gene expression analyses were performed to assess the expression of receptors activating the classical and non-canonical pathway, such as Fn14 and CD40, levels of NF-κB1/RelA and NF-κB2/RelB including its upstream signaling proteins, and expression of cytokines as target molecules of both pathways. In line with a higher expression of Fn14 and CD40, we detected an enhanced translocation of NF-κB1 and RelA as well as NF-κB2 and RelB into the nucleus accompanied by an increased gene expression of the NF-κB1-target cytokines Ccl20, Cxcl2, Ccl2, Cxcl1, IL-6, TNF-α, Cxcl10, and Ccl5, indicating an activation of the classical and non-canonical NF-κB pathway. Thereby, we provide, for the first time, in vivo evidence for an involvement of both NF-κB signaling pathways in renal pathophysiology of STEC-HUS.

X-Linked Immunodeficient Mice With No Functional Bruton's Tyrosine Kinase Are Protected From Sepsis-Induced Multiple Organ Failure.

We previously reported the Bruton's tyrosine kinase (BTK) inhibitors ibrutinib and acalabrutinib improve outcomes in a mouse model of polymicrobial sepsis. Now we show that genetic deficiency of the BTK gene alone in Xid mice confers protection against cardiac, renal, and liver injury in polymicrobial sepsis and reduces hyperimmune stimulation ("cytokine storm") induced by an overwhelming bacterial infection. Protection is due in part to enhanced bacterial phagocytosis in vivo, changes in lipid metabolism and decreased activation of NF-κB and the NLRP3 inflammasome. The inactivation of BTK leads to reduced innate immune cell recruitment and a phenotypic switch from M1 to M2 macrophages, aiding in the resolution of sepsis. We have also found that BTK expression in humans is increased in the blood of septic non-survivors, while lower expression is associated with survival from sepsis. Importantly no further reduction in organ damage, cytokine production, or changes in plasma metabolites is seen in Xid mice treated with the BTK inhibitor ibrutinib, demonstrating that the protective effects of BTK inhibitors in polymicrobial sepsis are mediated solely by inhibition of BTK and not by off-target effects of this class of drugs.

S1P lyase inhibition protects against sepsis by promoting disease tolerance via the S1P/S1PR3 axis.

BACKGROUND: One-third of all deaths in hospitals are caused by sepsis. Despite its demonstrated prevalence and high case fatality rate, antibiotics remain the only target-oriented treatment option currently available. Starting from results showing that low-dose anthracyclines protect against sepsis in mice, we sought to find new causative treatment options to improve sepsis outcomes. METHODS: Sepsis was induced in mice, and different treatment options were evaluated regarding cytokine and biomarker expression, lung epithelial cell permeability, autophagy induction, and survival benefit. Results were validated in cell culture experiments and correlated with patient samples. FINDINGS: Effective low-dose epirubicin treatment resulted in substantial downregulation of the sphingosine 1-phosphate (S1P) degrading enzyme S1P lyase (SPL). Consequent accumulation and secretion of S1P in lung parenchyma cells stimulated the S1P-receptor type 3 (S1PR3) and mitogen-activated protein kinases p38 and ERK, reducing tissue damage via increased disease tolerance. The protective effects of SPL inhibition were absent in S1PR3 deficient mice. Sepsis patients showed increased expression of SPL, stable expression of S1PR3, and increased levels of mucin-1 and surfactant protein D as indicators of lung damage. INTERPRETATION: Our work highlights a tissue-protective effect of SPL inhibition in sepsis due to activation of the S1P/S1PR3 axis and implies that SPL inhibitors and S1PR3 agonists might be potential therapeutics to protect against sepsis by increasing disease tolerance against infections. FUNDING: This study was supported by the Center for Sepsis Control and Care (CSCC), the German Research Foundation (DFG), RTG 1715 (to M. H. G. and I. R.) and the National Institutes of Health, Grant R01GM043880 (to S. S.).

Effect of Vitamin D on Peripheral Blood Mononuclear Cells from Patients with Psoriasis Vulgaris and Psoriatic Arthritis.

BACKGROUND: Psoriasis, a chronic skin disease with or without joint inflammation, has increased circulating proinflammatory cytokine levels. Vitamin D is involved in calcium homeostasis, bone formation, osteoclastogenesis and osteoclast activity, as well as regulation of immune response. We aimed to study osteoclast differentiation and cytokine secretion of peripheral blood mononuclear cells (PBMCs) from patients with psoriasis vulgaris and psoriatic arthritis, in response to 1,25(OH)2D3. METHODS: Serum levels of bone turnover markers were measured by ELISA in patients with psoriasis vulgaris and psoriatic arthritis, and healthy controls. PBMCs were isolated and cultured with or without RANKL/M-CSF and 1,25(OH)2D3. Osteoclast differentiation and cytokine secretion were assessed. RESULTS: Psoriatic arthritis patients had lower osteocalcin, as well as higher C-telopeptide of type I collagen and cathepsin K serum levels compared with psoriasis vulgaris patients and controls. RANKL/M-CSF-stimulated PBMCs from psoriatic arthritis patients produced higher proinflammatory cytokine levels and had a differential secretion profile in response to 1,25(OH)2D3, compared with psoriasis vulgaris and control PBMCs. CONCLUSIONS: Our data confirmed altered bone turnover in psoriatic arthritis patients, and demonstrated increased osteoclastogenic potential and proinflammatory cytokine secretion capacity of these PBMCs compared with psoriasis vulgaris and controls. 1,25(OH)2D3 abrogated these effects.

Low DEFB4 copy number and high systemic hBD-2 and IL-22 levels are associated with dermatophytosis.

Dermatophytes initiate dermatophytosis, but susceptibility to infection is dictated by host genetic factors, although the role of some of these-such as human beta-defensin 2 (hBD-2) genomic (DEFB4) copy number (CN) variation and its induction by IL-22-remains unclear. This was investigated in this cross-sectional study in 442 unrelated Caucasian subjects, including 195 healthy controls and 247 dermatophytosis patients who were divided into five subgroups according to clinical presentation. DNA samples were evaluated for DEFB4 CN variation by relative quantification using the comparative CT method, and serum hBD-2 and IL-22 levels were determined by ELISA. DEFB4 CN in patients was significantly lower and, except in the tinea cruris subgroup, serum hBD-2 levels were higher than in controls. The positive correlation between hBD-2 levels and DEFB4 CN observed in controls was not detected in patients, who also had higher serum IL-22 levels that were positively correlated with hBD-2 levels. Moreover, unlike in control subjects, the serum IL-22 level was negatively correlated with DEFB4 CN in patients. Taken together, these findings suggest an association between decreased DEFB4 CN, elevated serum hBD-2 and IL-22 levels, and dermatophytosis, underscoring a gene/cytokine interaction in the occurrence of this infection.

Native chromatographic sample preparation of serum, plasma and cerebrospinal fluid does not comprise a risk for proteolytic biomarker loss.

We recently developed a native multidimensional chromatographic method for serum and plasma fractionation for proteomic biomarker search. This method has several advantages:parallelization and automation, high reproducibility and proteome coverage, flexible dynamic range with respect to molecular weight and sample amount, optional enzymatic and immunological analytics additional to mass spectrometry, retaining metabolites, and information on complex formation, modification, and fragmentation of constituents. Nevertheless, native conditions have the probable risk of proteome alteration and biomarker loss by intrinsic proteinases. Hence, we tried to quantify here intrinsic proteolytic activity in native samples and fractions from serum, plasma and cerebrospinal fluid, as well as the effectiveness of intrinsic anti-proteinases during sample handling and preparation under our fractionation conditions. Therefore, we used several quantitative measures: (1) total proportion of intrinsic protein and peptide fractions, (2) azocasein hydrolysis and (3) mass spectrometric protein coverage and peptide numbers. To 1: In all non-fractionated specimens, neither decrease of protein concentration or molecular weight nor increase of peptide concentration was found after variable clotting or pre-incubation time. To 2: No azocasein hydrolysis was seen in these samples when prepared within a few hours at room temperature. Trypsin, when added in concentrations not higher than 0.85 µg/mL (0.04 µM), even was completely inhibited. Moreover, in native 1-D fractions no proteinase activity could be observed. To 3: Mass spectrometry confirmed that neither protein coverage nor peptide numbers differ significantly in 1-D or 2-D fractions after variable incubation time. These results suggest that intrinsic, native proteinase inhibitors potentially protect the proteomes considered, enabling "top-down" proteomic approaches under native conditions with serum, plasma and cerebrospinal fluid.