Macrophage Smad3 Protects the Infarcted Heart, Stimulating Phagocytosis and Regulating Inflammation.

RATIONALE: TGF (transforming growth factor)-ß is critically involved in myocardial injury, repair, and fibrosis, activating both Smad (small mothers against decapentaplegic)-dependent and non-Smad pathways. The in vivo role of TGF-ß signaling in regulation of macrophage function is poorly understood. We hypothesized that in the infarcted myocardium, activation of TGF-ß/Smad signaling in macrophages may regulate repair and remodeling. OBJECTIVE: To investigate the role of macrophage-specific TGF-ß Smad3 signaling in a mouse model of myocardial infarction and to dissect the mechanisms mediating Smad-dependent modulation of macrophage function. METHODS AND RESULTS: TGF-ßs markedly activated Smad3 in macrophages, without affecting Smad-independent pathways. Phagocytosis rapidly and directly activated macrophage Smad3, in the absence of active TGF-ß release. MyS3KO (myeloid cell-specific Smad3 knockout) mice had no baseline defects but exhibited increased late mortality and accentuated dilative postmyocardial infarction remodeling. Adverse outcome in infarcted MyS3KO mice was associated with perturbations in phagocytic activity, defective transition of macrophages to an anti-inflammatory phenotype, scar expansion, and accentuated apoptosis of border zone cardiomyocytes. In vitro, Smad3 null macrophages exhibited reduced expression of genes associated with eat-me signals, such as Mfge8 (milk fat globule-epidermal growth factor factor 8), and reduced capacity to produce the anti-inflammatory mediators IL (interleukin)-10 and TGF-ß1, and the angiogenic growth factor VEGF (vascular endothelial growth factor). Mfge8 partly rescued the phagocytic defect of Smad3 null macrophages, without affecting inflammatory activity. Impaired anti-inflammatory actions of Smad3 null macrophages were associated with marked attenuation of phagocytosis-induced PPAR (peroxisome proliferator-activated receptor) expression. MyS3KO mice had no significant alterations in microvascular density and interstitial fibrosis in remodeling myocardial segments. CONCLUSIONS: We demonstrate that Smad3 critically regulates function of infarct macrophages, by mediating acquisition of a phagocytic phenotype and by contributing to anti-inflammatory transition. Smad3-dependent actions in macrophages protect the infarcted heart from adverse remodeling.

Neuromuscular Complications of Programmed Cell Death-1 (PD-1) Inhibitors.

PURPOSE OF REVIEW: In recent years, immune checkpoint inhibitors have been increasingly used in patients with metastatic cancers with favorable oncological outcomes; however, there have also been increasing number of cancer survivors who have developed immune-related adverse events. Little is known about PD-1 inhibitor-associated neuromuscular complications. RECENT FINDINGS: Neuromuscular disorders are the most common neurological complication reported in PD-1 inhibitor-treated patients. Myasthenia gravis, immune-mediated myopathies, and Guillain-Barre syndrome are among commonly reported immune-related neuromuscular complications. HyperCKemia occurs frequently in patients with PD-1 inhibitor-associated myasthenia gravis, indicating coexisting myopathies or myocarditis. Oculobulbar weakness is a unique and common presentation of PD-1 inhibitor-associated immune-mediated myopathies with or without concomitant myasthenia gravis. High-dose steroid monotherapy may be associated with clinical deterioration in some patients with PD-1 inhibitor-associated myasthenia gravis, immune-mediated myopathies, or Guillain-Barre syndrome. PD-1 inhibitor-associated neuromuscular complications have some characteristic features compared to their idiopathic counterparts. Although steroid monotherapy is commonly used in non-neuromuscular autoimmune disorders triggered by anti-PD-1 therapy, this may lead to unfavorable outcomes in some patients with PD-1 inhibitor-associated neuromuscular complications.

Sex-Based Utilization of Guideline Recommended Statin Therapy and Cardiovascular Disease Outcomes: A Primary Prevention Healthcare Network Registry.

Background: In the US, women have similar cardiovascular death rates than men. Less is known about sex differences in statin use for primary prevention and associated atherosclerotic cardiovascular disease (ASCVD) outcomes. Methods: Statin prescriptions using electronic health records were examined in patients without ASCVD (myocardial infarction (MI), revascularization or ischemic stroke) between 2013-2019. Guideline-directed statin intensity (GDSI) at index and follow-up visits were compared among sexes across ASCVD risk groups, defined by pooled-cohort equation. Cox regression hazard ratios (HR) [95% CI] were calculated for statin use and outcomes (myocardial infarction, stroke/transient ischemic attack (TIA), and all-cause mortality) stratified by sex. Interaction terms (statin and sex) were applied. Results: Among 282,298 patients, (mean age ∼ 50 years) 17.1% women and 19.5% men were prescribed any statin at index visit. Time to GDSI was similar between sexes, but the proportion of high-risk women on GDSI at follow-ups was lower compared to high-risk men (2-years: 27.7 vs 32.0%, and 5-years: 47.2 vs 55.2%, p<0.05). When compared to GDSI, no statin use was associated with higher risk of MI and ischemic stroke/TIA amongst both sexes. High-risk women on GDSI had a lower risk of mortality (HR=1.39 [1.22-1.59]) versus men (HR=1.67 [1.50-1.86]) of similar risk (p value interaction=0.004). Conclusion: In a large contemporary healthcare system, there was underutilization of statins across both sexes in primary prevention. High-risk women were less likely to be initiated on GDSI compared with high-risk men. GDSI significantly improved the survival in both sexes regardless of ASCVD risk group. Future strategies to ensure continued use of GDSI, specifically among women, should be explored.

Sex-based utilization of guideline recommended statin therapy and cardiovascular disease outcomes: Data from a multisite healthcare network primary prevention cohort.

Background: In the US, women have similar cardiovascular death rates as men. However, less is known about sex differences in statin use for primary prevention and associated atherosclerotic cardiovascular disease (ASCVD) outcomes. Methods: Statin prescriptions using electronic health records were examined in patients without ASCVD (myocardial infarction (MI), revascularization or ischemic stroke) between 2013 and 2019. Guideline-directed statin intensity (GDSI) at index (at least moderate intensity, defined per pooled-cohort equation) and follow-up visits were compared between sexes across ASCVD risk groups, defined by the pooled-cohort equation. Cox regression hazard ratios were calculated for statin use and outcomes (myocardial infarction, stroke/transient ischemic attack (TIA), and all-cause mortality) stratified by sex. Interaction terms (statin and sex) were applied. Results: Among 282,298 patients, (mean age ∼ 50 years) 17.1 % women and 19.5 % men were prescribed any statin at index visit. Time to GDSI was similar between sexes, but the proportion of high-risk women on GDSI at follow-up were lower compared to high-risk men (2-years: 27.7 vs 32.0 %, and 5-years: 47.2 vs 55.2 %, p < 0.05). When compared to GDSI, no statin use was associated with higher risk of MI and ischemic stroke/TIA among both sexes. High-risk women on GDSI had a lower risk of mortality (HR=1.39 [1.22-1.59]) vs. men (HR=1.67 [1.50-1.86]) of similar risk (p value interaction=0.004). Conclusion: In a large contemporary healthcare system, there was underutilization of statins across both sexes in primary prevention. High-risk women were less likely to remain on GDSI compared to high-risk men. GDSI significantly improved the survival in both sexes regardless of ASCVD risk group. Future strategies to ensure continued use of GDSI, specifically among women, should be explored.

Lineage(-)Sca1+c-Kit(-)CD25+ cells are IL-33-responsive type 2 innate cells in the mouse bone marrow.

IL-33 promotes type 2 immune responses, both protective and pathogenic. Recently, targets of IL-33, including several newly discovered type 2 innate cells, have been characterized in the periphery. In this study, we report that bone marrow cells from wild-type C57BL/6 mice responded with IL-5 and IL-13 production when cultured with IL-33. IL-33 cultures of bone marrow cells from Rag1 KO and Kit(W-sh/W-sh) mice also responded similarly; hence, eliminating the possible contributions of T, B, and mast cells. Rather, intracellular staining revealed that the IL-5- and IL-13-positive cells display a marker profile consistent with the Lineage(-)Sca-1(+)c-Kit(-)CD25(+) (LSK(-)CD25(+)) cells, a bone marrow cell population of previously unknown function. Freshly isolated LSK(-)CD25(+) cells uniformly express ST2, the IL-33 receptor. In addition, culture of sorted LSK(-)CD25(+) cells showed that they indeed produce IL-5 and IL-13 when cultured with IL-33 plus IL-2 and IL-33 plus IL-7. Furthermore, i.p. injections of IL-33 or IL-25 into mice induced LSK(-)CD25(+) cells to expand, in both size and frequency, and to upregulate ST2 and α(4)ß(7) integrin, a mucosal homing marker. Thus, we identify the enigmatic bone marrow LSK(-)CD25(+) cells as IL-33 responsive, both in vitro and in vivo, with attributes similar to other type 2 innate cells described in peripheral tissues.

Investigation of the KIR4.1 potassium channel as a putative antigen in patients with multiple sclerosis: a comparative study.

BACKGROUND: Antibodies have been implicated in the pathogenicity of multiple sclerosis by findings of immunoglobulins in patients' CSF and often IgG and complement in lesions, and by a 2012 report that nearly half of patients' serum samples contain IgG specific for a glial potassium-channel, KIR4.1. We aimed to establish the frequency of KIR4.1-binding IgG in serum and CSF of patients with multiple sclerosis, and whether KIR4.1 immunoreactivity is retained or lost in demyelinating lesions. METHODS: Using ELISA with a KIR4.1 peptide, we tested archival serum from 229 population-based and 57 clinic-based patients with multiple sclerosis, 99 healthy controls, and 109 disease controls, and CSF from 25 patients with multiple sclerosis and 22 disease controls. We tested all CSF and serum samples from 50 of the clinic-based patients with multiple sclerosis on cells expressing functional KIR4.1, using cell-based immunofluorescence and immunoprecipitation (solubilised recombinant human KIR4.1). We assessed KIR4.1 immunoreactivity in archival brain samples from 15 patients with histopathologically confirmed multiple sclerosis (22 plaques [eight early active, eight inactive, and six remyelinated], 13 periplaque regions and eight normal-appearing white-matter and grey-matter regions) and from three controls with non-neurological diseases. FINDINGS: Three of 286 serum samples from patients with multiple sclerosis and two of 208 serum samples from controls showed KIR4.1 reactivity on ELISA; none of the CSF samples from patients or controls showed KIR4.1 reactivity. IgG in none of the 50 serum samples from clinic-based patients immunoprecipitated KIR4.1, but a commercial KIR4.1-specific control IgG did. By immunofluorescence, one of 50 serum samples from patients with multiple sclerosis yielded faint plasmalemmal staining on both KIR4.1-expressing and non-expressing cells; 16 bound faintly to intracellular components. In all cases, IgG binding was quenched by absorption with liver powder or lysates from non-transfected cells. Binding by the KIR4.1-specific control IgG was quenched only by lysates containing KIR4.1. IgG in none of the 25 CSF samples from patients with multiple sclerosis bound to KIR4.1-transfected cells. Glial KIR4.1 immunoreactivity was increased relative to expression in healthy control brain in all active demyelinating lesions, remyelinated lesions, and periplaque white matter regions. INTERPRETATION: We did not detect KIR4.1-specific IgG in serum or CSF from patients with multiple sclerosis or KIR4.1 loss from glia in multiple sclerosis lesions. Serological testing for KIR4.1-specific IgG is unlikely to aid diagnosis of multiple sclerosis. The target antigen of multiple sclerosis remains elusive. FUNDING: The National Institutes of Health, the National Multiple Sclerosis Society, and the Mayo Clinic Robert and Arlene Kogod Center on Aging.

2B4 utilizes ITAM-containing receptor complexes to initiate intracellular signaling and cytolysis.

2B4 is a member of the SLAM receptor family capable of activating NK cell cytotoxicity in the context of EBV infection. SAP (SLAM Associated Protein) deficiency causes defective signaling downstream of SLAM family receptors and high susceptibility to EBV. 2B4 costimulates natural cytotoxicity receptor (NCR) and TCR initiated signals to induce cellular cytotoxicity and cytokine release. The 2B4-SAP signal transduction pathway is not predicted to overlap with the TCR-ITAM pathway, although SAP is required for some TCR-induced signals. We therefore examined the functional relationship between SLAM family receptor 2B4 and ITAM-containing adaptor complexes. Removal of FcɛRIγ or CD3ζ-containing complexes, using genetically manipulated cell lines or siRNA specific suppression, significantly reduces 2B4-initiated functions in NK and T cells, respectively. Consistent with this relationship, Syk and ZAP-70 are capable of transducing 2B4 signals for calcium mobilization and cytolysis. Furthermore, ITAM-containing molecules constitutively associate with SAP. These results suggest a potential physical association between 2B4 and the ITAM receptor complexes that is required for 2B4-initiated signaling and cell-mediated killing.

Cutting edge: syntaxin 11 regulates lymphocyte-mediated secretion and cytotoxicity.

Little is known about the regulatory roles of specific soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in cytotoxic lymphocytes. Recent information suggests that mutations in the SNARE protein syntaxin 11 result in a form of familial hemophagocytic lymphohistiocytosis (FHL). Because genetic abnormalities in key granule components (e.g., perforin) or in regulators of secretion (e.g., Munc13-4) underlie the other identified forms of FHL, we assessed whether syntaxin 11 might also serve a related regulatory role. We determined that syntaxin 11 is expressed in NK cells and activated CTLs and is located in discrete membrane-associated structures in the cytoplasm. Enhanced expression of syntaxin 11 augments the secretion and killing of tumor targets, and suppression of syntaxin 11 expression inhibits these functions. Our data identify and characterize a role for syntaxin 11 in granule exocytosis and in the generation of cell-mediated killing. These results also provide new insights on the mechanisms of hemopoietic dysregulation in FHL.