Serum concentration of extracellular cold-inducible RNA-binding protein is associated with respiratory failure in COVID-19.

Uncontrolled release of damage-associated molecular patterns (DAMPs) is suggested to be a major trigger for the dysregulated host immune response that leads to severe COVID-19. Cold-inducible RNA-binding protein (CIRP), is a newly identified DAMP that aggravates inflammation and tissue injury, and induces respiratory failure in sepsis. Whether CIRP contributes to the pathogenesis of respiratory failure in COVID-19 has not yet been explored. Aim: To investigate if the concentration of extracellular CIRP (eCIRP) in serum associates with respiratory failure and lung involvement by chest computed tomography (CT) in COVID-19. Methods: Herein we report a prospective observational study of patients with COVID-19 included at two University Hospitals in Sweden between April 2020 and May 2021. Serum from hospitalized patients in Örebro (N=97) were used to assess the association between eCIRP and the level of respiratory support and its correlation with pulmonary involvement on chest CT and inflammatory biomarkers. A cohort of hospitalized and non-hospitalized patients from Umeå (N=78) was used as an external validation cohort. The severity of disease was defined according to the highest degree of respiratory support; mild disease (no oxygen), non-severe hypoxemia (conventional oxygen or high-flow nasal oxygen, HFNO <50% FiO2), and severe hypoxemia (HFNO ≥50% FiO2, mechanical ventilation). Unadjusted and adjusted linear regression was used to evaluate peak eCIRP day 0-4 in respect to severity, age, sex, Charlson comorbidity score, symptom duration, and BMI. Results: Peak eCIRP concentrations were higher in patients with severe hypoxemia and were independently associated with the degree of respiratory support in both cohorts (Örebro; p=0.01, Umeå; p<0.01). The degree of pulmonary involvement measured by CT correlated with eCIRP, rs=0.30, p<0.01 (n=97). Conclusion: High serum levels of eCIRP are associated with acute respiratory failure in COVID-19. Experimental studies are needed to determine if treatments targeting eCIRP reduces the risk of acute respiratory failure in COVID-19.

Sustained elevation of soluble B- and T- lymphocyte attenuator predicts long-term mortality in patients with bacteremia and sepsis.

Soluble B and T lymphocyte attenuator (sBTLA) has been shown to be associated with severity and outcome, in critically ill septic patients. We aimed to assess the dynamic expression of sBTLA, as a prognostic biomarker of long-term mortality in patients with bloodstream infection (BSI) and sepsis, and to evaluate its association with biomarkers indicative of inflammation and immune dysregulation. Secondarily, sBTLA was evaluated in association with severity and bacterial etiology. Patients with BSI (n = 108) were prospectively included, and serially sampled from admission to day 28. Blood and plasma donors (n = 31), sampled twice 28 days apart, served as controls. sBTLA concentration in plasma was determined with enzyme-linked immunosorbent assay. Associations between sBTLA on day 1-2 and 7, and mortality at 90 days and 1 year, were determined with unadjusted, and adjusted Cox regression. Differences related to severity was assessed with linear regression. Mixed model was used to assess sBTLA dynamics over time, and sBTLA associations with bacterial etiology and other biomarkers. sBTLA on day 1-2 and 7 was associated with mortality, in particular failure to normalize sBTLA by day 7 was associated with an increased risk of death before day 90, adjusted HR 17 (95% CI 1.8-160), and one year, adjusted HR 15 (95% CI 2.8-76). sBTLA was positively associated with CRP, and negatively with lymphocyte count. sBTLA on day 1-2 was not linearly associated with baseline SOFA score increase. High SOFA (≥4) was however associated with higher mean sBTLA than SOFA ≤3. sBTLA was not associated with bacterial etiology. We show that sustained elevation of sBTLA one week after hospital admission is associated with late mortality in patients with BSI and sepsis, and that sBTLA concentration is associated with CRP and decreased lymphocyte count. This suggests that sBTLA might be an indicator of sustained immune-dysregulation, and a prognostic tool in sepsis.

Patients With Microscopic Colitis Have Altered Levels of Inhibitory and Stimulatory Biomarkers in Colon Biopsies and Sera Compared to Non-inflamed Controls.

Introduction: Microscopic colitis (MC) is an inflammatory bowel condition with two subtypes, lymphocytic colitis (LC) and collagenous colitis (CC). Unlike patients with ulcerative colitis (UC) and non-inflamed individuals, MC patients have reduced risk of developing colorectal cancer, possibly due to increased immune surveillance in MC patients. Aim: To examine differences in levels of immunomodulatory molecules, including those involved in immune checkpoint mechanisms, in sera from patients with MC and in colonic biopsies from patients with MC and UC compared with controls. Methods: Using Luminex, 23 analytes (4-1BB, 4-1BBL, APRIL, BAFF, BTLA, CD27, CD28, CD80, CTLA-4, E-cadherin, Galectin-3, GITR, HVEM, IDO, IL-2Rα, LAG-3, MICA, MICB, PD-1, PD-L1, PD-L2, sCD40L and TIM-3) were studied in serum from patients with active MC (n = 35) and controls (n = 23), and in colonic biopsies from patients with active LC (n = 9), active CC (n = 16) and MC in histological remission (LC n = 6, CC n = 6), active UC (n = 15) and UC in remission (n = 12) and controls (n = 58). Results: In serum, IDO, PD-1, TIM-3, 4-1BB, CD27, and CD80 were decreased whereas 4-1BBL and IL-2Rα were increased in MC patients compared with controls. In contrast, in biopsies, levels of PD-L2 and 4-1BB were increased in MC and UC patients with active disease. Furthermore, in biopsies from CC and UC but not LC patients with active disease, CTLA-4, PD-1, APRIL, BAFF, and IL-2Rα were increased compared with controls. PD-L1 was increased in CC but not UC or LC patients. CD27 and TIM-3 were decreased in biopsies from MC patients in comparison to controls whereas levels of MICB were decreased in patients with active UC compared with controls. Conclusions: Compared with non-inflamed controls, levels of soluble and membrane-bound immunomodulatory molecules were systemically and locally altered in MC and UC patients, with most analytes being decreased in serum but enhanced in colonic biopsies. These findings contribute to knowledge about checkpoint molecules and their role as biomarkers in MC and may also contribute to knowledge about possible mechanisms behind the seemingly protective effects of MC against colorectal cancer.

A combination of the activation marker CD86 and the immune checkpoint marker B and T lymphocyte attenuator (BTLA) indicates a putative permissive activation state of B cell subtypes in healthy blood donors independent of age and sex.

BACKGROUND: The use of anti-B cell based therapies in immune-mediated diseases targeting general B cell markers or molecules important for B cell function has increased the clinical needs of monitoring B cell subpopulations. RESULTS: We analyzed the expression profile of cell surface markers CD86 and B and T lymphocyte attenuator (BTLA) in B cell subtypes using flow cytometry, including naïve, transitional, switched memory, non-switched memory and double-negative memory B cells and plasmablasts, and investigated the dependence of age and sex in a healthy adult blood donor population. The switched memory B cell subtype displayed a divergent expression of the markers, with increased CD86 and decreased BTLA as compared to non-switched and double negative memory cells, as well as compared to naïve B cells. Plasmablasts expressed highly increased CD86 compared to all other subtypes and a decreased expression of BTLA compared to naïve cells, but still higher compared to the memory cell populations. Transitional B cells had CD86 and BTLA expression similar to the other naïve cells. CONCLUSIONS: We show divergent expression of CD86 and BTLA in memory cells and plasmablasts compared to naïve B cells independent of age and sex. Furthermore, a similarly divergent difference of expression pattern was seen between the memory cell subtypes, altogether indicating that the combination of CD86 and BTLA might be markers for a permissive activation state. We suggest the combination of CD86 and BTLA expression on B cell subtypes as a potentially important tool in monitoring the status of B cell subtypes before and after treatments influencing the B cell compartment.

Plasma concentrations of secretory leukocyte protease inhibitor (SLPI) differ depending on etiology and severity in community-onset bloodstream infection.

The severity of bloodstream infections (BSI) depends on pathogen, source, and host factors. Secretory leukocyte protease inhibitor (SLPI) counteracts tissue damage, balances inflammation, and is increased in pneumonia and sepsis. We aimed to evaluate whether SLPI production differs depending on etiology, disease severity, and sex in BSI and to correlate SLPI with markers of inflammation and immunosuppression. Of the adult patients with BSI, 109 were included and sampled repeatedly, from hospital admission through day 28. Controls (blood donors) were sampled twice. SLPI in plasma was measured with enzyme-linked immunosorbent assay (ELISA) technique. Streptococcus pneumoniae and Staphylococcus aureus etiology were associated with higher SLPI than Escherichia coli on days 1-2 and 3. On day 1-2, subjects with sepsis had higher SLPI concentrations than those with non-septic BSI. Pneumonia was associated with higher SLPI than a non-pulmonary source of infection. SLPI co-varied with inflammatory markers. SLPI concentrations did not differ with regard to sex in the full cohort, but men with pneumonia had higher SLPI than women on day 1-2. S. pneumoniae and S. aureus BSI were associated with higher SLPI, when compared to E. coli. Severity and pneumonia, as well as male sex in the pneumonia sub-cohort, were factors independently associated with higher SLPI.

Oligoclonal T-cell Receptor Repertoire in Colonic Biopsies of Patients with Microscopic Colitis and Ulcerative Colitis.

BACKGROUND: Microscopic colitis (MC), comprising collagenous colitis (CC) and lymphocytic colitis (LC), is a type of variation of inflammatory bowel diseases. Local T-cell infiltration in the mucosa plays a major role in MC immunopathology. METHODS: To understand diversity and clonality of infiltrating T cells, we analyzed the T-cell receptor beta (TCRß) chains in colonic biopsies of MC, ulcerative colitis (UC), and their remission counterparts (CC/LC-HR [histological remission] or UC-R [remission]) compared with patients with noninflamed colons using next-generation sequencing. RESULTS: Compared with controls and patients with CC, patients with LC had significantly lower diversity with significantly lower evenness and richness in TCRVß-Jß gene segments. Similarly, patients with LC-HR had lower diversity because of significantly lower TCRVß-Jß clone richness. Patients with UC and UC-R showed significantly higher diversity and richness. Univariate and multivariate analyses were performed to identify TCRVß-Jß gene segments differentiating disease types from controls or their remission counterparts. Patients with LC were discriminated from controls by 12 clones and from patients with CC by 8 clones. Neither univariate nor multivariate analyses showed significance for patients with CC or CC-HR compared with controls. Patients with UC and UC-R had 16 and 14 discriminating clones, respectively, compared with controls. CONCLUSIONS: Altogether, patients with MC and UC showed an oligoclonal TCRß distribution. TCRVß-Jß clone types and their diversity were distinctive between patients with CC and LC, as well as for patients with UC, suggesting different pathophysiological mechanisms according to disease type and stage. This study suggests that CC and LC are different entities because of differences in immunoregulatory responses, as mirrored by their T-cell repertoire.

Soluble B and T Lymphocyte Attenuator Correlates to Disease Severity in Sepsis and High Levels Are Associated with an Increased Risk of Mortality.

INTRODUCTION AND AIMS: B- and T-lymphocyte Attenuator (BTLA), Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and Programmed Death 1 (PD-1) are co-inhibitory receptors that regulate T cell activation. In the present study of ICU-treated patients we measured plasma concentrations of their soluble isoforms, with the aim to evaluate their potential as sepsis biomarkers and utility as prognostic indicators. METHODS: 101 patients with sepsis, 28 patients with non-infectious critical illness (ICU controls) and 31 blood donors (healthy controls, HC) were included in the study. Plasma concentrations of soluble BTLA (sBTLA), CTLA-4 (sCTLA-4) and PD-1 (sPD-1) were measured with ELISA in serial blood samples. Comparisons were made with Mann-Whitney U test and correlations were assessed with Spearman's Rank correlation test. Cox proportional hazard models, with sBTLA and sPD-1 as fixed and sBTLA as time-varying covariates, were used to determine association with 28-day mortality. RESULTS: sBTLA levels were significantly higher in the sepsis cohort (median 14 ng/mL, IQR 8-29) compared to ICU controls (9 ng/mL, IQR 5-26, p = 0.048) and HC (2.9 ng/mL, IQR 0.9-9.1, p<0.01), and correlated to SOFA score. sBTLA levels were higher in 28 day sepsis non-survivors than in survivors (baseline median 28 ng/mL, IQR 13-41 vs 13 ng/mL, IQR 8-23, p = 0.04). After adjustment for age and comorbidities, the relative risk of 28 day mortality was nearly 5-fold higher in sepsis patients with a baseline sBTLA > 21 ng/mL, compared to those with a level below this threshold. sBTLA was even more associated with mortality in the time-varying analysis. sPD-1 levels were lower in the sepsis cohort compared to HC but not compared to ICU controls and were not associated with mortality. sCTLA-4 was detectable in only one subject. CONCLUSION: Plasma concentrations of soluble BTLA were increased early in sepsis/septic shock and correlated to severity of disease. A baseline concentration >21ng/mL was associated with a poor prognosis.

Reduced IL-37 Production Increases Spontaneous Chemokine Expressions in Colon Epithelial Cells.

BACKGROUND AND AIM: Microscopic colitis, comprising collagenous colitis and lymphocytic colitis, is a common cause of chronic diarrhea. Previously, we showed enhanced chemokine productions in microscopic colitis patients, indicating dysregulated immune cell chemotaxis in the immunopathogenesis. We also showed decreased mRNA of IL-37, mainly regarded as an anti-inflammatory cytokine, in the colonic mucosa of these patients, potentially an important factor for the chronicity of the colitis. Our aim in this study was to understand the possible role of IL-37 in chemokine production using a cell line model. METHODS: A colon epithelial cell line, T84, was stimulated with the TLR5 ligand flagellin. IL-37 protein production was reduced 20% using the CRISPR/Cas9 system, and the changes in chemokine mRNA and protein expressions were compared to cells transfected with empty plasmid. RESULTS: The 20% reduction in IL-37 protein levels spontaneously increased CCL5, CXCL8, CXCL10, and CXCL11 mRNA and protein expressions. CCL2 mRNA and protein levels were enhanced upon TLR5 stimulation. CCL3, CCL20, and CX3CL1 mRNA expressions were increased either spontaneously or following TLR5 stimulation, whereas CCL4 and CCL22 mRNA expressions were significantly decreased. CONCLUSIONS: Even a minor decrease in the ability of colon epithelial cells to produce IL-37 results in altered chemokine expression, mainly an increase in the production of several chemokines. Our results indicate that a decreased IL-37 expression by colon epithelial cells may be an important factor for increasing the recruitment of immune cells and subsequently developing microscopic colitis.

Enhanced levels of chemokines and their receptors in the colon of microscopic colitis patients indicate mixed immune cell recruitment.

Microscopic colitis (MC), comprising collagenous colitis (CC) and lymphocytic colitis (LC), is a common cause of chronic diarrhea. Various immune cell infiltrations in the epithelium and lamina propria are seen in MC immunopathology. We compared gene and protein expressions of different immune cell attracting chemokines and their receptors in colon biopsies from MC patients in active disease or histopathological remission (CC/LC-HR) with controls, using qRT-PCR and Luminex, respectively. CC and LC patients with active disease demonstrated a mixed chemokine profile with significantly enhanced gene and/or protein expressions of the chemokines CCL2, CCL3, CCL4, CCL5, CCL7, CCL22, CXCL8, CXCL9, CXCL10, CXCL11, and CX3CL1 and the receptors CCR2, CCR3, CCR4, CXCR1, CXCR2, and CX3CR1. Enhanced chemokine/chemokine receptor gene and protein levels in LC-HR patients were similar to LC patients, whereas CC-HR patients demonstrated almost normalized levels. These findings expand the current understanding of the involvement of various immune cells in MC immunopathology and endorse chemokines as potential diagnostic markers as well as therapeutic candidates. Moreover, this study further supports the hypothesis that CC and LC are two different entities due to differences in their immunoregulatory responses.

An in vitro model to evaluate the impact of the soluble factors from the colonic mucosa of collagenous colitis patients on T cells: enhanced production of IL-17A and IL-10 from peripheral CD4⁺ T cells.

Soluble factors from intestinal mucosal cells contribute to immune homeostasis in the gut. We have established an in vitro model to investigate the regulatory role of soluble factors from inflamed intestinal mucosa of collagenous colitis (CC) patients in the differentiation of T cells. Peripheral blood CD4(+) T cells from healthy donors were polyclonally activated in the presence of conditioned medium (CM) generated from denuded biopsies (DNB) or isolated lamina propria mononuclear cells (LPMCs) from mucosal biopsies from CC patients compared to noninflamed controls, to determine proliferation and secretion of cytokines involved in T-cell differentiation. Compared to controls, we observed significantly increased production of the proinflammatory cytokines IFN-γ, IL-17A, IL-6, and IL-1ß and the anti-inflammatory cytokines IL-4 and IL-10 in the presence of CC-DNB-CM. The most pronounced effect of CC-LPMC-CM on peripheral CD4(+) T cells was a trend towards increased production of IL-17A and IL-10. A trend towards reduced inhibition of T-cell proliferation was noted in the presence of CC-DNB-CM. In conclusion, our in vitro model reveals implications of soluble factors from CC colonic mucosa on peripheral T cells, enhancing their production of both pro- and anti-inflammatory cytokines.

Differential expression of interleukin-1/Toll-like receptor signaling regulators in microscopic and ulcerative colitis.

AIM: To investigate Toll-like receptor (TLR) signaling regulators in microscopic and ulcerative colitis patients. METHODS: Total RNA and microRNA were isolated from fresh frozen colonic biopsies of non-inflamed controls and patients with active or in-remission collagenous colitis (CC), lymphocytic colitis (LC), or ulcerative colitis (UC). We compared expressions of interleukin-1 receptor-associated kinase (IRAK)-2, IRAK-M, interleukin (IL)-37, microRNA (miR)-146a, miR-155, and miR-21 using quantitative real time reverse transcription polymerase chain reaction. RESULTS: IRAK-M expression was increased in LC patients with active disease in histopathological remission (LC-HR; P = 0.02) and UC patients (P = 0.01), but no differences in IRAK-2 expression were detected compared to controls. miR-146a, -155 and -21 expressions were increased in LC-HR (P = 0.04, 0.07, and 0.004) and UC (P = 0.02, 0.04 and 0.03) patients. miR-146a and miR-21 expressions were significantly enhanced in UC patients compared to UC remission (UC-R; P = 0.01 and 0.04). Likewise, active CC patients showed significantly increased expression of miR-155 (P = 0.003) and miR-21 (P = 0.006). IL-37 expression was decreased in both CC (P = 0.03) and LC (P = 0.04) patients with a similar trend in UC patients but not statistically significant, whilst it was increased in UC-R patients compared to controls (P = 0.02) and active UC (P = 0.001). CONCLUSION: The identification of differentially expressed miRNAs, IL-37, and IRAK-M suggests different pathophysiologic mechanisms in various disease stages in LC, CC, and UC.

International recommendations for the assessment of autoantibodies to cellular antigens referred to as anti-nuclear antibodies.

Anti-nuclear antibodies (ANA) are fundamental for the diagnosis of autoimmune diseases, and have been determined by indirect immunofluorescence assay (IIFA) for decades. As the demand for ANA testing increased, alternative techniques were developed challenging the classic IIFA. These alternative platforms differ in their antigen profiles, sensitivity and specificity, raising uncertainties regarding standardisation and interpretation of incongruent results. Therefore, an international group of experts has created recommendations for ANA testing by different methods. Two groups of experts participated in this initiative. The European autoimmunity standardization initiative representing 15 European countries and the International Union of Immunologic Societies/World Health Organization/Arthritis Foundation/Centers for Disease Control and Prevention autoantibody standardising committee. A three-step process followed by a Delphi exercise with closed voting was applied. Twenty-five recommendations for determining ANA (1-13), anti-double stranded DNA antibodies (14-18), specific antibodies (19-23) and validation of methods (24-25) were created. Significant differences between experts were observed regarding recommendations 24-25 (p<0.03). Here, we formulated recommendations for the assessment and interpretation of ANA and associated antibodies. Notably, the roles of IIFA as a reference method, and the importance of defining nuclear and cytoplasmic staining, were emphasised, while the need to incorporate alternative automated methods was acknowledged. Various approaches to overcome discrepancies between methods were suggested of which an improved bench-to-bedside communication is of the utmost importance. These recommendations are based on current knowledge and can enable harmonisation of local algorithms for testing and evaluation of ANA and related autoantibodies. Last but not least, new more appropriate terminologies have been suggested.

Antimicrobial peptide plasma concentrations in patients with community-acquired pneumonia.

BACKGROUND: Community-acquired pneumonia (CAP) is a common and potentially life-threatening infection. Innate immunity is the first line of defence, and antimicrobial peptides (AMPs) produced by white blood cells and at epithelial barriers participate by killing microorganisms and neutralizing bacterial toxins. We wanted to investigate whether concentrations of AMPs (1) are increased in CAP, (2) predict the clinical outcome, and (3) differ depending on the causative microbe. METHODS: Plasma concentrations of AMPs were measured using an enzyme-linked immunosorbent assay in 89 patients with CAP, 21 patients with non-respiratory tract infections (non-RTI), and 63 healthy control subjects. RESULTS: In subjects with CAP, mean plasma concentrations of secretory leukocyte protease inhibitor (SLPI) and bactericidal/ permeability-increasing protein (BPI) were significantly higher than in healthy control subjects (85 vs 45 ng/ml, p < 0.001 and 48 vs 10 ng/ml, p < 0.001, respectively), but less markedly increased in patients with non-RTI (68 ng/ml, p = 0.06 and 41 ng/ml, p = 0.43). LL-37 and human neutrophil peptides 1-3 (HNP 1-3) levels were not increased in subjects with CAP. Levels of BPI and SLPI did not correlate to severity of disease, and AMP levels did not differ depending on the causative agent. Interestingly, male subjects with CAP displayed increased concentrations of SLPI compared to females. This was not observed in subjects with non-RTI and healthy control subjects. CONCLUSIONS: Subjects with CAP showed increased plasma concentrations of SLPI and BPI compared to healthy control subjects. The finding of higher SLPI levels in male subjects with CAP implies that there are sex-dependent immunological differences in SLPI turnover.

Elution of antitransglutaminase antibodies from duodenal biopsies: a novel approach in the diagnosis of celiac disease.

Celiac disease (CeD) is a disease more prevalent and multisymptomatic than was earlier recognized. Whereas prompt initiation of gluten-free diet (GFD) is beneficial in relieving the symptoms, an accurate CeD diagnosis is necessary also to avoid years of restricted diet on uncertain grounds. We propose a new diagnostic method, based on elution of deposited antibodies against transglutaminase (anti-tTG) from duodenal biopsies in patients with symptoms and screening serology analyses suggestive of CeD. The eluates were analyzed in a Phadia 250 fluoroimmunoassay, demonstrating elevated concentrations of anti-tTG in CeD patients, corresponding to serology and histopathology findings. In one case histology was inconclusive, displaying only unspecific inflammation, but eluted anti-tTG was positive. This patient has clinically improved following GFD. We conclude that our novel method represents a new tool in the diagnostic work up in CeD. The detection of deposited anti-tTG at the site of inflammation appears to provide a high sensitivity and specificity using a technique that is quick, simple and reliable. Further studies are needed for optimization and elucidation of suitable applications for this elution method.

IL-1 receptor-associated kinase M downregulates DSS-induced colitis.

BACKGROUND: Ulcerative colitis is associated with increased colon permeability resulting in bacterial translocation into the lamina propria. We investigate the importance of the Toll-like receptor (TLR) regulating protein IL-1 receptor-associated kinase M (IRAK-M) using the erosive dextran sulfate sodium (DSS)-induced model of colitis. METHODS: IRAK-M-competent and -incompetent mice were treated with 3% DSS for 5 days followed by 2 days of regular drinking water. Clinical signs of disease were followed for 7 days. At day 7 the mice were sacrificed and plasma and tissue were collected for histopathological examination and analyses of the production of cytokines and chemokines as well as expression of T-cell transcription factors. RESULTS: At day 7 IRAK-M-deficient mice display a reduced total body weight (77.1 ± 2.1 versus 88.5 ± 2.0, *P = 0.002) and an increased macroscopical (2.7 ± 0.2 versus 1.6 ± 0.1, *P = 0.002) and histopathological (6.0 ± 0 versus 3.3 ± 0.5, *P = < 0.001) colon score compared to wildtype mice. Furthermore, IRAK-M-deficient mice have increased colon mRNA expression of proinflammatory cytokines and increased tumor necrosis factor concentrations (41.1 ± 13.5 versus 12.8 ± 2.0 pg/mL, *P = 0.010) in plasma. CONCLUSIONS: This is the first report examining the role of IRAK-M in colitis. We find that IRAK-M is of critical importance in downregulating induction and progression of DSS colitis, and thereby suggesting that IRAK-M might be a target for future interventional therapies.

Gender dependent importance of IRAK-1 in dextran sulfate sodium induced colitis.

Toll-like receptor (TLR) signaling is important for the induction of pro-inflammatory cytokines and interferon (IFN)-inducible genes in response to bacterial and viral challenge. Interleukin-1 receptor-associated kinase-1 (IRAK-1) is a signaling kinase situated downstream of the adapter protein myeloid differentiation factor 88 (MyD88) in the TLR intracellular signaling cascade and is required for normal signal transduction through this pathway. We investigated the importance of IRAK-1 in intestinal inflammation by using the dextran sulfate sodium (DSS)-colitis model. We show that IRAK-1 deficient mice are protected against systemic signs of inflammation, i.e., weight loss and spleen enlargement compared to wild-type controls irrespective of gender. However, IRAK-1(-/y) males but not IRAK-1(-/-) females display significant protection against colitis and thymic atrophy compared to wild-type mice. Our results indicate a gender specific effect of IRAK-1 in the DSS-induced colitis, an interesting finding since the Irak-1 gene is located on the X-chromosome and several inflammatory diseases have a gender dependent incidence.

Mature-onset obesity in interleukin-1 receptor I knockout mice.

Interleukin-1 (IL-1) is a major mediator of inflammation that exerts its biological activities through the IL-1 type I receptor (IL-1RI). The body weights of IL-1RI(-/-) mice of both sexes started to deviate from those of wild-type mice at 5-6 months of age and were 20% higher at 9 months of age. Visceral and subcutaneous fat mass, measured by dual-energy X-ray absorptiometry and magnetic resonance imaging, was markedly (1.5- to 2.5-fold) increased. Lean body mass and crown-rump length were also slightly (11 and 5%, respectively) increased, as was serum IGF-I. Obese IL-1RI(-/-) mice were insulin resistant, as evidenced by hyperinsulinemia, decreased glucose tolerance, and insulin sensitivity. To elucidate the mechanisms for the development of obesity, young pre-obese IL-1RI(-/-) mice were investigated. They showed decreased suppression of body weight and food intake in response to systemic leptin treatment. The decreased leptin responsiveness was even more pronounced in older obese animals. Moreover, spontaneous locomotor activity and fat utilization, as measured by respiratory quotient, were decreased in pre-obese IL-1RI(-/-) mice. In conclusion, lack of IL-1RI-mediated biological activity causes mature-onset obesity. This obese phenotype is preceded by decreased leptin sensitivity, fat utilization, and locomotor activity.

IL-1 receptor-associated kinase 1 mediates protection against Staphylococcus aureus infection.

The interleukin-1 receptor-associated kinase-1 (IRAK-1) mediates signal transduction from Toll-like/IL-1/IL-18 receptors. Though a critical protective role against Staphylococcus aureus infection has been previously attributed to myeloid differentiation factor 88 (MyD88) and IRAK-4, both also involved in TLR/IL-1/IL-18 signaling, the role of IRAK-1 is unknown. IRAK-1-deficient (IRAK-1-/-) and wild-type mice were inoculated i.v. with 2 x 10(7) or 1 x 10(6) S. aureus per mouse to evaluate the role of IRAK-1 in S. aureus sepsis. Since IRAK-1 transduces IL-1R signals, IL-1R-/- mice were also included in experiments. IRAK-1-/- mice are susceptible to a high dose of S. aureus compared to wild-type controls. In contrast to the high mortality and extensive weight loss seen in IL-1R-deficient mice in response to 1 x 10(6) S. aureus, IRAK-1-/- mice are resistant to this low dose of S. aureus. Thus IRAK-1 plays an important role in the host response to staphylococcal sepsis.

Enhanced pro-inflammatory cytokine production in Galphai2-deficient mice on colitis prone and colitis resistant 129Sv genetic backgrounds.

Mice deficient in G-protein subunit alphai2 develop colitis closely resembling human ulcerative colitis when raised on 129SvEv background. When backcrossing the Galphai2-deficiency into a 129SvJBom genetic background, surprisingly, mice did not develop colitis. In vitro stimulation of splenocytes with formalin-killed Staphylococcus aureus resulted in significantly increased production of interleukin-1beta, tumor necrosis factor, and interleukin-12p40 in Galphai2(-/-) as compared to control mice. The enhanced production of pro-inflammatory cytokines was seen in colitis prone as well as in colitis resistant genetic background. A similar outcome was seen upon stimulation with toxic shock syndrome toxin-1, a T cell superantigen, except that Galphai2(-/-) colitis resistant 129SvJBom splenocytes did not show increased production of IL-12p40 as compared to their controls.

T-box transcription-factor-deficient mice display increased joint pathology and failure of infection control during staphylococcal arthritis.

To study the impact of T-box transcription factor (T-bet) on initiation and progression of Staphylococcus aureus sepsis and arthritis, T-bet-deficient mice (T-bet(-/-)) and their wild-type controls (T-bet(+/+)) were intravenously inoculated with 8 x 10(6) S. aureus. Already 48 h after inoculation of S. aureus, T-bet-deficient mice displayed increased frequency (62% versus 19%, P = 0.002) as well as severity of arthritis compared with wild-type controls. The bacterial counts were significantly increased in T-bet(-/-) mice compared with T-bet(+/+) as measured in kidneys 72 h after the inoculation (4.3 +/- 1.8 x 10(7) versus 3.2 +/- 3.2 x 10(6) colony-forming units (CFU); P = 0.003). As expected, T-bet-deficient mice displayed significantly decreased production of IFN-gamma (10-15-fold) at 24 and 72 h after bacterial inoculation compared with wild-type mice. Interestingly, in the absence of T-bet, serum IL-4 was decreased at 24 h. IL-6 did not differ at early stage of infection but was sixfold increased in T-bet(-/-) mice over T-bet(+/+) animals at 72 h postinoculation. Ten days after the inoculation, T-bet(-/-) mice still displayed significantly more pronounced weight loss and increased serum IL-6 levels, probably due to increased bacterial burden compared with T-bet(+/+) mice. The cumulative mortality was 19% in T-bet mice (5/27) and 0% (0/27) in control animals (P = 0.05). In conclusion, T-bet plays an important role in early response to S. aureus infection, protecting against bacterial accumulation, cachexia and septic death. Furthermore T-bet downregulates joint inflammation in the early phase of disease.