IL11-mediated stromal cell activation may not be the master regulator of pro-fibrotic signaling downstream of TGFß.

Fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF) and systemic scleroderma (SSc), are commonly associated with high morbidity and mortality, thereby representing a significant unmet medical need. Interleukin 11 (IL11)-mediated cell activation has been identified as a central mechanism for promoting fibrosis downstream of TGFß. IL11 signaling has recently been reported to promote fibroblast-to-myofibroblast transition, thus leading to various pro-fibrotic phenotypic changes. We confirmed increased mRNA expression of IL11 and IL11Rα in fibrotic diseases by OMICs approaches and in situ hybridization. However, the vital role of IL11 as a driver for fibrosis was not recapitulated. While induction of IL11 secretion was observed downstream of TGFß signaling in human lung fibroblasts and epithelial cells, the cellular responses induced by IL11 was quantitatively and qualitatively inferior to that of TGFß at the transcriptional and translational levels. IL11 blocking antibodies inhibited IL11Rα-proximal STAT3 activation but failed to block TGFß-induced profibrotic signals. In summary, our results challenge the concept of IL11 blockade as a strategy for providing transformative treatment for fibrosis.

Outcomes of hematopoietic stem cell gene therapy for Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is a rare X-linked disorder characterized by combined immunodeficiency, eczema, microthrombocytopenia, autoimmunity, and lymphoid malignancies. Gene therapy (GT) to modify autologous CD34+ cells is an emerging alternative treatment with advantages over standard allogeneic hematopoietic stem cell transplantation for patients who lack well-matched donors, avoiding graft-versus-host-disease. We report the outcomes of a phase 1/2 clinical trial in which 5 patients with severe WAS underwent GT using a self-inactivating lentiviral vector expressing the human WAS complementary DNA under the control of a 1.6-kB fragment of the autologous promoter after busulfan and fludarabine conditioning. All patients were alive and well with sustained multilineage vector gene marking (median follow-up: 7.6 years). Clinical improvement of eczema, infections, and bleeding diathesis was universal. Immune function was consistently improved despite subphysiologic levels of transgenic WAS protein expression. Improvements in platelet count and cytoskeletal function in myeloid cells were most prominent in patients with high vector copy number in the transduced product. Two patients with a history of autoimmunity had flares of autoimmunity after GT, despite similar percentages of WAS protein-expressing cells and gene marking to those without autoimmunity. Patients with flares of autoimmunity demonstrated poor numerical recovery of T cells and regulatory T cells (Tregs), interleukin-10-producing regulatory B cells (Bregs), and transitional B cells. Thus, recovery of the Breg compartment, along with Tregs appears to be protective against development of autoimmunity after GT. These results indicate that clinical and laboratory manifestations of WAS are improved with GT with an acceptable safety profile. This trial is registered at clinicaltrials.gov as #NCT01410825.

Dealer-customer partnership in rice production demonstration: Assessment of private extension system in Bangladesh.

Traditional public extension worker-farmer cooperation in rice production demonstration is not working efficiently, therefore, private partnership-based demonstration has been attempted to introduce as its alternative very recently involving dealer-customer farmer. The study evaluated the private extension services rendered through dealer-customer farmer cooperation in Bangladesh. Thirty-three rice seed dealers and ninety-two customer farmers formed the samples for the study. Face-to-face interviews were employed as a quantitative method while focus group discussion was used as a qualitative method in the present study. Involving in the private rice production demonstration approach, customer farmers indicated high profit, greater involvement in decision-making, and improved marketing skills as the major advantages; while the dealers stressed the benefit received by the small farmers, improvement in their decision-making capacity and increased local rice production. However, the slow distribution of inputs during the production period was a weakness in the arrangement, which was mostly because of the dealers' lack of understanding of the customer farmers' needs. The private extension system being a new concept in the country may be observed over a period and gradually extended to the nooks and crannies of the country.

Utilizing a reductionist model to study host-microbe interactions in intestinal inflammation.

BACKGROUND: The gut microbiome is altered in patients with inflammatory bowel disease, yet how these alterations contribute to intestinal inflammation is poorly understood. Murine models have demonstrated the importance of the microbiome in colitis since colitis fails to develop in many genetically susceptible animal models when re-derived into germ-free environments. We have previously shown that Wiskott-Aldrich syndrome protein (WASP)-deficient mice (Was-/-) develop spontaneous colitis, similar to human patients with loss-of-function mutations in WAS. Furthermore, we showed that the development of colitis in Was-/- mice is Helicobacter dependent. Here, we utilized a reductionist model coupled with multi-omics approaches to study the role of host-microbe interactions in intestinal inflammation. RESULTS: Was-/- mice colonized with both altered Schaedler flora (ASF) and Helicobacter developed colitis, while those colonized with either ASF or Helicobacter alone did not. In Was-/- mice, Helicobacter relative abundance was positively correlated with fecal lipocalin-2 (LCN2), a marker of intestinal inflammation. In contrast, WT mice colonized with ASF and Helicobacter were free of inflammation and strikingly, Helicobacter relative abundance was negatively correlated with LCN2. In Was-/- colons, bacteria breach the mucus layer, and the mucosal relative abundance of ASF457 Mucispirillum schaedleri was positively correlated with fecal LCN2. Meta-transcriptomic analyses revealed that ASF457 had higher expression of genes predicted to enhance fitness and immunogenicity in Was-/- compared to WT mice. In contrast, ASF519 Parabacteroides goldsteinii's relative abundance was negatively correlated with LCN2 in Was-/- mice, and transcriptional analyses showed lower expression of genes predicted to facilitate stress adaptation by ASF519 in Was-/-compared to WT mice. CONCLUSIONS: These studies indicate that the effect of a microbe on the immune system can be context dependent, with the same bacteria eliciting a tolerogenic response under homeostatic conditions but promoting inflammation in immune-dysregulated hosts. Furthermore, in inflamed environments, some bacteria up-regulate genes that enhance their fitness and immunogenicity, while other bacteria are less able to adapt and decrease in abundance. These findings highlight the importance of studying host-microbe interactions in different contexts and considering how the transcriptional profile and fitness of bacteria may change in different hosts when developing microbiota-based therapeutics. Video abstract.

Intracellular MLCK1 diversion reverses barrier loss to restore mucosal homeostasis.

Epithelial barrier loss is a driver of intestinal and systemic diseases. Myosin light chain kinase (MLCK) is a key effector of barrier dysfunction and a potential therapeutic target, but enzymatic inhibition has unacceptable toxicity. Here, we show that a unique domain within the MLCK splice variant MLCK1 directs perijunctional actomyosin ring (PAMR) recruitment. Using the domain structure and multiple screens, we identify a domain-binding small molecule (divertin) that blocks MLCK1 recruitment without inhibiting enzymatic function. Divertin blocks acute, tumor necrosis factor (TNF)-induced MLCK1 recruitment as well as downstream myosin light chain (MLC) phosphorylation, barrier loss, and diarrhea in vitro and in vivo. Divertin corrects barrier dysfunction and prevents disease development and progression in experimental inflammatory bowel disease. Beyond applications of divertin in gastrointestinal disease, this general approach to enzymatic inhibition by preventing access to specific subcellular sites provides a new paradigm for safely and precisely targeting individual properties of enzymes with multiple functions.

STAT1 signaling shields T cells from NK cell-mediated cytotoxicity.

The JAK-STAT pathway critically regulates T-cell differentiation, and STAT1 is postulated to regulate several immune-mediated diseases by inducing proinflammatory subsets. Here we show that STAT1 enables CD4+ T-cell-mediated intestinal inflammation by protecting them from natural killer (NK) cell-mediated elimination. Stat1-/- T cells fail to expand and establish colitis in lymphopenic mice. This defect is not fully recapitulated by the combinatorial loss of type I and II IFN signaling. Mechanistically, Stat1-/- T cells have reduced expression of Nlrc5 and multiple MHC class I molecules that serve to protect cells from NK cell-mediated killing. Consequently, the depletion of NK cells significantly rescues the survival and spontaneous proliferation of Stat1-/- T cells, and restores their ability to induce colitis in adoptive transfer mouse models. Stat1-/- mice however have normal CD4+ T cell numbers as innate STAT1 signaling is required for their elimination. Overall, our findings reveal a critical perspective on JAK-STAT1 signaling that might apply to multiple inflammatory diseases.

Constitutive activation of WASp in X-linked neutropenia renders neutrophils hyperactive.

Congenital neutropenia is characterized by low absolute neutrophil numbers in blood, leading to recurrent bacterial infections, and patients often require life-long granulocyte CSF (G-CSF) support. X-linked neutropenia (XLN) is caused by gain-of-function mutations in the actin regulator Wiskott-Aldrich syndrome protein (WASp). To understand the pathophysiology in XLN and the role of WASp in neutrophils, we here examined XLN patients and 2 XLN mouse models. XLN patients had reduced myelopoiesis and extremely low blood neutrophil number. However, their neutrophils had a hyperactive phenotype and were present in normal numbers in XLN patient saliva. Murine XLN neutrophils were hyperactivated, with increased actin dynamics and migration into tissues. We provide molecular evidence that the hyperactivity of XLN neutrophils is caused by WASp in a constitutively open conformation due to contingent phosphorylation of the critical tyrosine-293 and plasma membrane localization. This renders WASp activity less dependent on regulation by PI3K. Our data show that the amplitude of WASp activity inside a cell could be enhanced by cell-surface receptor signaling even in the context in which WASp is already in an active conformation. Moreover, these data categorize XLN as an atypical congenital neutropenia in which constitutive activation of WASp in tissue neutrophils compensates for reduced myelopoiesis.

WASP-mediated regulation of anti-inflammatory macrophages is IL-10 dependent and is critical for intestinal homeostasis.

Mutations in Wiskott-Aldrich syndrome protein (WASP) cause autoimmune sequelae including colitis. Yet, how WASP mediates mucosal homeostasis is not fully understood. Here we show that WASP-mediated regulation of anti-inflammatory macrophages is critical for mucosal homeostasis and immune tolerance. The generation and function of anti-inflammatory macrophages are defective in both human and mice in the absence of WASP. Expression of WASP specifically in macrophages, but not in dendritic cells, is critical for regulation of colitis development. Importantly, transfer of WT anti-inflammatory macrophages prevents the development of colitis. DOCK8-deficient macrophages phenocopy the altered macrophage properties associated with WASP deficiency. Mechanistically, we show that both WASP and DOCK8 regulates macrophage function by modulating IL-10-dependent STAT3 phosphorylation. Overall, our study indicates that anti-inflammatory macrophage function and mucosal immune tolerance require both WASP and DOCK8, and that IL-10 signalling modulates a WASP-DOCK8 complex.

Temperature dependent infrared nano-imaging of La0.67Sr0.33MnO3 thin film.

We investigate the temperature dependence of infrared properties at nanometer length scales in La0.67Sr0.33MnO3 (LSMO) thin film with a thickness of 47 unit cells grown on SrTiO3 substrate. The infrared nano-imaging experiments were performed using a near-field optical microscope in conjunction with a variable temperature heating stage. The near-field infrared data is consistent with the bulk of the LSMO film undergoing the thermally-driven non-percolative second-order transition from a metallic, ferromagnetic phase to an insulating, paramagnetic phase. We find persistent infrared contrast on the nanoscale that is independent of temperature and which we attribute to two novel phases with different conductivities coexisting in the vicinity of the film-substrate interface. These two coexisting phases at the film-substrate interface do not undergo the metal-insulator transition (MIT) and hence are different from the metallic, ferromagnetic and insulating, paramagnetic phases in the bulk of the film. At temperatures approaching the nominal MIT temperature, repeated scans of the same microscopic area at constant temperature reveal bimodal fluctuation of the near-field infrared amplitude. We interpret this phenomenon as slow, critical fluctuations of the conductivity in the bulk of the LSMO film.

Enhanced TH17 Responses in Patients with IL10 Receptor Deficiency and Infantile-onset IBD.

BACKGROUND: IL10 receptor (IL10R) deficiency causes severe infantile-onset inflammatory bowel disease. Intact IL10R-dependent signals have been shown to be important for innate and adaptive immune cell functions in mice. We have previously reported a key role of IL10 in the generation and function of human anti-inflammatory macrophages. Independent of innate immune cell defects, the aim of the current study was to determine the role of IL10R signaling in regulating human CD4 T-cell function. METHODS: Peripheral blood mononuclear cells and intestinal biopsies cells were collected from IL10/IL10R-deficient patients and controls. Frequencies of CD4 T-cell subsets, naive T-cell proliferation, regulatory T cell (Treg)-mediated suppression, and Treg and TH17 generation were determined by flow cytometry. Transcriptional profiling was performed by NanoString and quantitative real-time polymerase chain reaction. RNA in situ hybridization was used to determine the quantities of various transcripts in intestinal mucosa. RESULTS: Analysis of 16 IL10- and IL10R-deficient patients demonstrated similar frequencies of peripheral blood and intestinal Tregs, compared with control subjects. In addition, in vitro Treg suppression of CD4 T-cell proliferation and generation of Treg were not dependent on IL10R signaling. However, IL10R-deficient T naive cells exhibited higher proliferative capacity, a strong TH17 signature, and an increase in polarization toward TH17 cells, compared with controls. Moreover, the frequency of TH17 cells was increased in the colon and ileum of IL10R-deficient patients. Finally, we show that stimulation of IL10R-deficient Tregs in the presence of IL1ß leads to enhanced production of IL17A. CONCLUSIONS: IL10R signaling regulates TH17 polarization and T-cell proliferation in humans but is not required for the generation and in vitro suppression of Tregs. Therapies targeting the TH17 axis might be beneficial for IL10- and IL10R-deficient patients as a bridge to allogeneic hematopoietic stem cell transplantation.

Macrophage dysfunction initiates colitis during weaning of infant mice lacking the interleukin-10 receptor.

Infants with defects in the interleukin 10 receptor (IL10R) develop very early onset inflammatory bowel disease. Whether IL10R regulates lamina propria macrophage function during infant development in mice and whether macrophage-intrinsic IL10R signaling is required to prevent colitis in infancy is unknown. Here we show that although signs of colitis are absent in IL10R-deficient mice during the first two weeks of life, intestinal inflammation and macrophage dysfunction begin during the third week of life, concomitant with weaning and accompanying diversification of the intestinal microbiota. However, IL10R did not directly regulate the microbial ecology during infant development. Interestingly, macrophage depletion with clodronate inhibited the development of colitis, while the absence of IL10R specifically on macrophages sensitized infant mice to the development of colitis. These results indicate that IL10R-mediated regulation of macrophage function during the early postnatal period is indispensable for preventing the development of murine colitis.

ADAMTS13 Deficiency Worsens Colitis and Exogenous ADAMTS13 Administration Decreases Colitis Severity in Mice.

BACKGROUND: Inflammatory bowel disease (IBD) affects 1.6 million people in the United States. IBD is associated with an increased risk of thrombosis, which rises with disease activity. The pathogenesis of IBD and its increased thrombotic risk is not completely understood. Ultra large von Willebrand factor (ULVWF) multimers are secreted from activated endothelium, leading to recruitment of platelets and leukocytes. A disintegrin and metalloproteinase with thrombospondin type I repeats motif 13 (ADAMTS13) cleaves highly adhesive ULVWF into smaller, less bioactive, multimers, releasing them into circulation. Mice deficient in ADAMTS13 (ADAMTS13-/-) have heightened inflammatory and thrombotic responses. OBJECTIVES: We hypothesized that upon colitis induction, ADAMTS13-/- mice would have more severe symptoms compared with wild-type (WT) mice, and rhADAMTS13 administration to mice with colitis would improve their condition. RESULTS: Dextran sodium sulfate-induced colitis was worse in ADAMTS13-/- mice than WT. ADAMTS13-/- showed increased weight loss, worse anemia, and increased clinical and histologic colitis severity, compared with WT mice. ADAMTS13-/- mice had increased VWF release, with accumulation at inflamed colonic sites. Also, the majority of mice showed one or more submucosal colonic thrombi. ADAMTS13 deficiency worsened colitis and propagated intestinal inflammation, most likely through increased platelet-leukocyte recruitment by VWF. Treatment of WT mice with rhA-DAMTS13 decreased colitis severity without worsening anemia. Additionally, several immune-mediated chronic murine colitis models, and inflamed colon tissue specimens from IBD patients, showed increased VWF release at inflamed sites, suggesting a generalizability of our findings. CONCLUSION: Measuring VWF/ADAMTS13 levels could have clinical utility. When applicable, the administration of ADAMTS13, in addition to primary treatment, may improve outcomes for IBD patients.

Interleukin 1ß Mediates Intestinal Inflammation in Mice and Patients With Interleukin 10 Receptor Deficiency.

Interleukin 10 receptor (IL10R)-deficient mice develop spontaneous colitis and, similarly, patients with loss-of-function mutations in IL10R develop severe infant-onset inflammatory bowel disease. Loss of IL10R signaling in mouse and human macrophages is associated with increased production of interleukin 1ß. We demonstrated that innate immune production of IL1ß mediates colitis in IL10R-deficient mice. Transfer of Il1r1-/- CD4+ T cells into Rag1-/-/Il10rb-/- mice reduced the severity of their colitis (compared to mice that received CD4+ T cells that express IL1R), accompanied by decreased production of interferon gamma, tumor necrosis factor-α, and IL17A. In macrophages from mice without disruption of IL10R signaling or from healthy humans (controls), incubation with IL10 reduced canonical activation of the inflammasome and production of IL1ß through transcriptional and post-translational regulation of NLRP3. Lipopolysaccharide and adenosine triphosphate stimulation of macrophages from Il10rb-/- mice or IL10R-deficient patients resulted in increased production of IL1ß. Moreover, in human IL10R-deficient macrophages, lipopolysaccharide stimulation alone triggered IL1ß secretion via non-canonical, caspase 8-dependent activation of the inflammasome. We treated 2 IL10R-deficient patients with severe and treatment-refractory infant-onset inflammatory bowel disease with the IL1-receptor antagonist anakinra. Both patients had marked clinical, endoscopic, and histologic responses after 4-7 weeks. This treatment served as successful bridge to allogeneic hematopoietic stem cell transplantation in 1 patient. Our findings indicate that loss of IL10 signaling leads to intestinal inflammation, at least in part, through increased production of IL1 by innate immune cells, leading to activation of CD4+ T cells. Agents that block IL1 signaling might be used to treat patients with inflammatory bowel disease resulting from IL10R deficiency.

A study on cognitive decline with respect to metabolic syndrome and inflammation in elderly Indians.

BACKGROUND: This study was undertaken to find out if metabolic syndrome (MetS) in the elderly was associated with cognitive decline and also if this association was modified by the presence of inflammation. MATERIALS AND METHODS: 100 patients more than 60 years of age were divided into 2 groups of 50 each and were age and sex matched. Group 1 and 2 had patients with and without MetS, respectively. The individual components of MetS were measured in each patient. Cognitive decline was measured by Modified Mini-Mental Score (3MS) of Teng. Inflammation was measured by high-sensitivity C-reactive protein (hs-CRP). RESULTS: Fasting hyperglycemia was the most common component of MetS (60% of group 1). The mean serum hs-CRP in patients of group 1 was 6.56 ± 9.72 while that in the patients of group 2 was 1.95 ± 1.93. In the group-1, 36% (n = 18) patients were having a decreased 3MS, whereas in group-2, 22% (n = 11) were having a decreased 3MS. MetS was associated with an odd's ratio of 1.99 for developing cognitive decline. 3MS had a negative correlation with hs-CRP values. Regression analysis showed a significant association of hs-CRP and MetS with cognitive decline in the elderly population. CONCLUSION: Cognitive decline in the elderly is associated with the presence of inflammation and MetS. Hence, early identification of the high-risk groups may offer benefit by disease course modification and better caregiving.

Interleukin-10 receptor signaling in innate immune cells regulates mucosal immune tolerance and anti-inflammatory macrophage function.

Intact interleukin-10 receptor (IL-10R) signaling on effector and T regulatory (Treg) cells are each independently required to maintain immune tolerance. Here we show that IL-10 sensing by innate immune cells, independent of its effects on T cells, was critical for regulating mucosal homeostasis. Following wild-type (WT) CD4(+) T cell transfer, Rag2(-/-)Il10rb(-/-) mice developed severe colitis in association with profound defects in generation and function of Treg cells. Moreover, loss of IL-10R signaling impaired the generation and function of anti-inflammatory intestinal and bone-marrow-derived macrophages and their ability to secrete IL-10. Importantly, transfer of WT but not Il10rb(-/-) anti-inflammatory macrophages ameliorated colitis induction by WT CD4(+) T cells in Rag2(-/-)Il10rb(-/-) mice. Similar alterations in the generation and function of anti-inflammatory macrophages were observed in IL-10R-deficient patients with very early onset inflammatory bowel disease. Collectively, our studies define innate immune IL-10R signaling as a key factor regulating mucosal immune homeostasis in mice and humans.

Interleukin 10 receptor signaling: master regulator of intestinal mucosal homeostasis in mice and humans.

Interleukin 10 (IL10) is a key anti-inflammatory cytokine that can inhibit proinflammatory responses of both innate and adaptive immune cells. An association between IL10 and intestinal mucosal homeostasis became clear with the discovery that IL10 and IL10 receptor (IL10R)-deficient mice develop spontaneous intestinal inflammation. Similarly, patients with deleterious mutations in IL10, IL10RA, or IL10RB present with severe enterocolitis within the first months of life. Here, we review recent findings on how IL10- and IL10R-dependent signaling modulates innate and adaptive immune responses in the murine gastrointestinal tract, with implications of their role in the prevention of inflammatory bowel disease (IBD). In addition, we discuss the impact of IL10 and IL10R signaling defects in humans and their relationship to very early-onset IBD (VEO-IBD).

A study on pulmonary complications of systemic sclerosis in eastern India.

AIM: This study was undertaken to find out the characteristics of clinical, radiological and functional changes affecting the respiratory system in patients with systemic sclerosis (SSc) from eastern India, and the association of these characteristics with pulmonary hypertension. METHODS: This was a cross-sectional, observational study involving 46 patients. Other than the routine tests, anti-nuclear antibody (ANA), spirometry, diffusing capacity of lung for carbon monoxide (DLCO) measurement, chest radiograph, high-resolution computed tomography (HRCT) of thorax, 6-minute walk test and echocardiography were done. RESULTS: Out of a total of 46 patients, 27 patients had diffuse cutaneous SSc (dcSSc) and 19 had limited cuteaneous SSc (lcSSc). Eleven patients had pulmonary hypertension. The HRCT revealed diffuse parenchymal lung disease (DPLD) in 32 (65%) cases. The ANA was positive in 83% cases. Anti-Scl70 was found in 41% of patients with dcSSc and anti-centromere antibody was found in 47% of patients with lcSSc. Spirometry revealed restrictive pattern in 30 patients; 9 had obstruction; and the rest were normal. The DLCO was abnormal in 38 patients. A strong correlation was found between reduction in DLCO and pulmonary artery systolic pressure (PASP). Also, a strong association was observed between a drop of > 4% in oxygen saturation on 6-minute walk test and presence of pulmonary arterial hypertension (PAH). CONCLUSIONS: Majority of the patients with SSc had restrictive lung disease with abnormal DLCO and features resembling non-specific interstitial pneumonia. Nucleolar ANA was predominantly found in patients having PAH. Presence of DPLD had a negative association with presence of anti-centromere antibody. Reduction in DLCO and a fall of > 4% in oxygen saturation on 6-minute walk test may be used as predictors of PAH in asymptomatic individuals.