Reduction in parvalbumin-positive interneurons and inhibitory input in the cortex of mice with experimental autoimmune encephalomyelitis

In multiple sclerosis (MS), inflammation leads to damage of central nervous system myelin and axons. Previous studies have postulated impaired GABA transmission in MS, and recent postmortem analysis has shown that GABAergic parvalbumin (PV)-positive interneurons are decreased in the primary motor cortex (M1) of patients with MS. In this report, we present evidence for the loss of a specific population of GABAergic interneurons in the experimental autoimmune encephalomyelitis mouse model of MS. Using experimental autoimmune encephalomyelitis, we evaluated the distribution of both PV-positive interneurons and of the inhibitory presynaptic input in the M1 of experimental autoimmune encephalomyelitis and control mice. Our results demonstrate a specific decrease in the number of PV-positive interneurons in the M1 of mice with experimental autoimmune encephalomyelitis. We detected a significant reduction in the number of PV-positive interneurons in the layers II and III of the M1 of diseased mice, while there was no difference in the number of calretinin (CR)-positive cells between animals with experimental autoimmune encephalomyelitis and control animals. Moreover, we observed a significant reduction in the inhibitory presynaptic input in the M1 of treated mice. These changes were specific for the mice with elevated clinical score, while they were not detectable in the mice with low clinical score. Our results support the hypothesis that reinforcing the action of the GABAergic network may represent a therapeutic alternative to limit the progression of the neuronal damage in MS patients.     

Differential Muscle Hypertrophy Is Associated with Satellite Cell Numbers and Akt Pathway Activation Following Activin Type IIB Receptor Inhibition in Mtm1 p.R69C Mice

X-linked myotubular myopathy is a congenital myopathy caused by deficiency of myotubularin. Patients often present with severe perinatal weakness, requiring mechanical ventilation to prevent death from respiratory failure. We recently reported that an activin receptor type IIB inhibitor produced hypertrophy of type 2b myofibers and modest increases of strength and life span in the severely myopathic Mtm1δ4 mouse model of X-linked myotubular myopathy. We have now performed a similar study in the less severely symptomatic Mtm1 p.R69C mouse in hopes of finding greater treatment efficacy. Activin receptor type IIB inhibitor treatment of Mtm1 p.R69C animals produced behavioral and histological evidence of hypertrophy in gastrocnemius muscles but not in quadriceps or triceps. The ability of the muscles to respond to activin receptor type IIB inhibitor treatment correlated with treatment-induced increases in satellite cell number and several muscle-specific abnormalities of hypertrophic signaling. Treatment-responsive Mtm1 p.R69C gastrocnemius muscles displayed lower levels of phosphorylated ribosomal protein S6 and higher levels of phosphorylated eukaryotic elongation factor 2 kinase than were observed in Mtm1 p.R69C quadriceps muscle or in muscles from wild-type littermates. Hypertrophy in the Mtm1 p.R69C gastrocnemius muscle was associated with increased levels of phosphorylated ribosomal protein S6. Our findings indicate that muscle-, fiber type-, and mutation-specific factors affect the response to hypertrophic therapies that will be important to assess in future therapeutic trials.X-linked myotubular myopathy (XLMTM) is a severe form of congenital myopathy with an estimated incidence of 1:50,000 male births that most often presents with severe perinatal weakness and respiratory failure.^1,2 Many patients with XLMTM die within the first year of life despite the use of mechanical ventilation, and no treatments approved by the Food and Drug Administration are available. XLMTM is caused by mutations in the gene that encodes myotubularin (MTM1), which is a phosphoinositide phosphatase thought to be involved in endosomal trafficking, cytoskeletal organization, apoptosis, and/or maintenance of the sarcoplasmic reticulum/T-tubular system within myofibers.^3–8 Muscle biopsies from patients with XLMTM display excessively small fibers with increased numbers of fibers that contain central nuclei and central aggregation of organelles.⁹ Although the number of centrally nucleated fibers bears little relationship to a patient''s prognosis, there is a clear correlation between the degree of fiber smallness at birth and the severity of the patients'' disease.¹⁰ Two murine models of myotubularin deficiency are used, the severely symptomatic Mtm1δ4 (also referred to as Mtm1 knockout in prior studies^3,11,12) and the moderately symptomatic Mtm1 p.R69C mice,¹³ both of which display weakness and myofiber smallness and similar pathology to that seen in XLMTM.Because of the relationship between myofiber size and symptomatic severity in patients with XLMTM and in Mtm1δ4 mice, we had previously hypothesized that correction of myofiber smallness in myotubularin deficiency would greatly improve strength. Inhibitors of myostatin or nonfunctional decoys of its receptor, the activin type IIB receptor (ActRIIB), can be used to inhibit this negative regulator of myofiber size, leading to myofiber hypertrophy. Myostatin binds to (and signals through) the ActRIIB to activate the transforming growth factor-β pathway, which prevents progression through the cell cycle and down-regulates several key processes related to myofiber hypertrophy.^14,15 We recently reported a trial of ActRIIB-mFC in Mtm1δ4 mice, which produced 17% extension of life span, with transient increases in weight, forelimb grip strength, myofiber size, and myofiber hypertrophy restricted to type 2b myofibers in Mtm1δ4 animals.¹² Interestingly, ActRIIB-mFc produces hypertrophy in all muscle fiber types in wild-type (WT) mice,^12,16 which suggests that myotubularin deficiency interferes with the activation of hypertrophic pathways in oxidative fibers.We hypothesized that the transience of the therapeutic effects observed in treated Mtm1δ4 mice may have been related to the severity of the disease, so we have now repeated this study in the less severely affected Mtm1 p.R69C mouse.¹³ Surprisingly, treatment of Mtm1 p.R69C mice did not produce significant increases in animal weight or grip strength, and treatment-induced myofiber hypertrophy was only observed in the Mtm1 p.R69C gastrocnemius muscles. The ability of these muscles to respond to ActRIIB-mFC treatment correlated with treatment-induced increases in satellite cell number and several muscle-specific abnormalities of hypertrophic signaling. The main difference between treatment-responsive (gastrocnemius) and treatment-resistant (quadriceps) muscles in Mtm1 p.R69C mice was related to low levels of phosphorylated ribosomal protein 6 (p-rpS6) and high levels of eukaryotic elongation factor 2 kinase (eEF2K) in the treatment-responsive gastrocnemius muscle that were not observed in other Mtm1 p.R69C muscles or in WT mice. rpS6 and eEF2K are terminal signaling molecules of the insulinlike growth factor-1/Akt and extracellular signal-related kinase (ERK) pathways that are involved in the fine-tuning of global protein synthesis, with a role in the determination of cell size that remains unclear (reviewed in Meyuhas¹⁷). Our findings indicate that the response to hypertrophic agents does not always correlate with activities of known hypertrophic pathways, such as the Akt pathway, but unexpectedly varies both by muscle type and fiber type and in XLMTM is affected by the nature of the Mtm1 mutation. These results highlight that there is much we still do not understand about the control of muscle size and emphasize the importance of evaluating multiple muscle and fiber types in future trials of hypertrophic therapies.     

Evidence that Meningeal Mast Cells Can Worsen Stroke Pathology in Mice

Stroke is the leading cause of adult disability and the fourth most common cause of death in the United States. Inflammation is thought to play an important role in stroke pathology, but the factors that promote inflammation in this setting remain to be fully defined. An understudied but important factor is the role of meningeal-located immune cells in modulating brain pathology. Although different immune cells traffic through meningeal vessels en route to the brain, mature mast cells do not circulate but are resident in the meninges. With the use of genetic and cell transfer approaches in mice, we identified evidence that meningeal mast cells can importantly contribute to the key features of stroke pathology, including infiltration of granulocytes and activated macrophages, brain swelling, and infarct size. We also obtained evidence that two mast cell-derived products, interleukin-6 and, to a lesser extent, chemokine (C-C motif) ligand 7, can contribute to stroke pathology. These findings indicate a novel role for mast cells in the meninges, the membranes that envelop the brain, as potential gatekeepers for modulating brain inflammation and pathology after stroke.Stroke, the leading cause of adult disability and the fourth most common cause of death in the Unites States,^1,2 occurs when there is insufficient blood flow to the brain, and the resultant injury initiates a cascade of inflammatory events, including immune cell infiltration into the brain.^3–5 This post-stroke inflammation is a critical determinant of damage and recovery after stroke; understanding the interplay between the immune system and the brain after stroke holds much promise for therapeutic intervention.^4–7 However, successfully exploiting this therapeutic potential requires a detailed understanding of the interplay between the immune system and the brain after stroke.⁴An understudied but important aspect of this interplay is the role of meningeal-located immune cells in modulating brain pathology. The meninges have long been recognized as an anatomical barrier that protects the central nervous system (CNS). However, accumulating evidence suggests that the meninges are important for communication between the CNS and immune system during health and disease.^8–10 All blood vessels pass through the meningeal subarachnoid space before entering the brain, and this vascular connection and the close proximity of the meninges to the underlying parenchymal nervous tissue make them ideally located to act as a gatekeeper to modulate immune cell trafficking to the CNS. To support this gatekeeper function is evidence that the meninges modulate brain infiltration of T cells, neutrophils, and monocytes during meningitis and autoimmune conditions,^11–14 with immune cells observed in some instances accumulating in the meninges before they infiltrate into the parenchyma.^11,13Emerging evidence suggests that the actions of immune cells resident in the meninges are important for this gatekeeper function.^11,12,15 Mast cells (MCs), best known as proinflammatory effector cells, can play critical roles in the development of inflammation in many disease settings.^16–18 MCs reside in high numbers within the meninges, but their function in this site has not been fully investigated in stroke pathology. Unlike most immune cells, mature MCs do not circulate in the blood but are long-term residents of tissues, often in perivascular locations, and can rapidly perform their functions in situ. CNS MCs are found in the brain parenchyma and the meninges of rodents and humans.¹⁸ It has been proposed that brain parenchymal MCs can enhance brain neutrophil numbers after stroke and can exacerbate stroke pathology.^19–24 However, much of the evidence to support such conclusions is indirect. For example, some of the studies that implicate MCs in stroke pathology used pharmacologic approaches to interfere with MC activation,^19,20,22 but such drugs can have effects on other cell types.²⁵ Moreover, the role of the meningeal MCs in modulating post-stroke inflammation and pathology is unknown. Finally, little is understood about which among the many MC-derived mediators may be important in stroke pathology.^17,26To address these questions, we used genetic and cell transfer approaches to study the role of MCs in the pathology of ischemic stroke in mice. Specifically, we tested a c-kit–mutant mouse model (ie, WBB6F₁-Kit^W/W-v mice) which is profoundly MC deficient and can be repaired of this deficiency by engraftment of in vitro-derived MCs from wild-type (WT) mice. This MC knock-in approach enables the MC-dependent effects in the mutant mice to be separated from effects due to other abnormalities associated with their mutation,^11,17,26,27 because only the MC deficiency is repaired by MC engraftment. Furthermore, one can investigate the mechanisms by which MCs influence stroke pathology by engrafting MCs from transgenic mice that lack specific MC-associated products. We also tested our newly described Cpa3-Cre; Mcl-1^fl/fl mice, in which MC (and basophil) numbers are reduced constitutively via Cre-mediated depletion of the anti-apoptotic factor, myeloid cell leukemia sequence 1 (Mcl-1), in the affected lineages.²⁸ Cpa3-Cre; Mcl-1^fl/fl mice lack the other abnormalities associated with the c-kit mutations in WBB6F₁-Kit^W/W-v mice.²⁸With the use of these in vivo models, we identified meningeal MCs as important contributors to key features of stroke pathology, including increased numbers of brain granulocytes and activated macrophages, brain swelling, and infarct size. We also obtained evidence that two potentially proinflammatory MC-derived products, IL-6 and, to a lesser extent, chemokine (C-C motif) ligand 7 (CCL7), can contribute to pathology in this setting.     

Interleukin-3-Deficient Mice Have Increased Resistance to Blood-Stage Malaria

The contribution of interleukin-3 (IL-3), a hematopoietic growth factor and immunoregulatory cytokine, to resistance to blood-stage malaria was investigated by infecting IL-3-deficient (knockout [KO]) mice with Plasmodium berghei NK65. Male IL-3 KO mice, but not female mice, were more resistant to infection than wild-type (WT) mice, as evidenced by lower peak parasitemia and prolonged survival. Both male and female IL-3 KO mice had increased splenomegaly and were more anemic than corresponding WT mice. Anemia was compensated for by an increase in bone marrow and splenic erythropoiesis in IL-3 KO mice, as evidenced by higher levels of erythroid progenitors. Plasma levels of gamma interferon (IFN-γ) and CXCL9 (monokine induced by IFN-γ [MIG]) were found to be significantly reduced in IL-3 KO mice during early stages of infection. In contrast, granulocyte colony-stimulating factor (G-CSF) levels were significantly higher, and the percentage of peripheral blood neutrophils lower, in infected IL-3 KO mice than in WT counterparts. Overall, our results indicate that IL-3 plays a critical role in suppressing protective immunity to P. berghei NK65 infection and that it is involved in inhibiting the development of splenomegaly, anemia, and erythropoiesis. IL-3 also influences IFN-γ, CXCL9, and G-CSF production in response to infection. The abnormal responses seen in infected IL-3 KO mice may be due to the lack of IL-3 during development, to the lack of IL-3 in the infected mature mice, or to both.     

Yersinia pseudotuberculosis Efficiently Escapes Polymorphonuclear Neutrophils during Early Infection

The human-pathogenic species of the Gram-negative genus Yersinia preferentially target and inactivate cells of the innate immune defense, suggesting that this is a critical step by which these bacteria avoid elimination and cause disease. In this study, bacterial interactions with dendritic cells, macrophages, and polymorphonuclear neutrophils (PMNs) in intestinal lymphoid tissues during early Yersinia pseudotuberculosis infection were analyzed. Wild-type bacteria were shown to interact mainly with dendritic cells, but not with PMNs, on day 1 postinfection, while avirulent yopH and yopE mutants interacted with PMNs as well as with dendritic cells. To unravel the role of PMNs during the early phase of infection, we depleted mice of PMNs by using an anti-Ly6G antibody, after which we could see more-efficient initial colonization by the wild-type strain as well as by yopH, yopE, and yopK mutants on day 1 postinfection. Dissemination of yopH, yopE, and yopK mutants from the intestinal compartments to mesenteric lymph nodes was faster in PMN-depleted mice than in undepleted mice, emphasizing the importance of effective targeting of PMNs by these Yersinia outer proteins (Yops). In conclusion, escape from interaction with PMNs due to the action of YopH, YopE, and YopK is a key feature of pathogenic Yersinia species that allows colonization and effective dissemination.     

Role of Pht Proteins in Attachment of Streptococcus pneumoniae to Respiratory Epithelial Cells

Pneumococcal adherence to mucosal surfaces is a critical step in nasopharyngeal colonization, but so far few pneumococcal adhesins involved in the interaction with host cells have been identified. PhtA, PhtB, PhtD, and PhtE are conserved pneumococcal surface proteins that have proven promising as vaccine candidates. One suggested virulence function of Pht proteins is to mediate adherence at the respiratory mucosa. In this study, we assessed the role of Pht proteins in pneumococcal binding to respiratory epithelial cells. Pneumococci were incubated with human nasopharyngeal epithelial cells (Detroit-562) and lung epithelial cells (A549 and NCI-H292), and the proportion of bound bacteria was measured by plating viable counts. Strains R36A (unencapsulated), D39 (serotype 2), 43 (serotype 3), 4-CDC (serotype 4), and 2737 (serotype 19F) with one or more of the four homologous Pht proteins deleted were compared with their wild-type counterparts. Also, the effect of anti-PhtD antibodies on the adherence of strain 2737 to the respiratory epithelial cells was studied. Our results suggest that Pht proteins play a role in pneumococcal adhesion to the respiratory epithelium. We also found that antibody to PhtD is able to inhibit bacterial attachment to the cells, suggesting that antibodies against PhtD present at mucosal surfaces might protect from pneumococcal attachment and subsequent colonization. However, the relative significance of Pht proteins to the ability of pneumococci to bind in vitro to epithelial cells depends on the genetic background and the capsular serotype of the strain.     

Phosphoinositide substrates of myotubularin affect voltage-activated Ca2+ release in skeletal muscle

Skeletal muscle excitation–contraction (E–C) coupling is altered in several models of phosphatidylinositol phosphate (PtdInsP) phosphatase deficiency and ryanodine receptor activity measured in vitro was reported to be affected by certain PtdInsPs, thus prompting investigation of the physiological role of PtdInsPs in E–C coupling. We measured intracellular Ca²⁺ transients in voltage-clamped mouse muscle fibres microinjected with a solution containing a PtdInsP substrate (PtdIns(3,5)P ₂ or PtdIns(3)P) or product (PtdIns(5)P or PtdIns) of the myotubularin phosphatase MTM1. No significant change was observed in the presence of either PtdIns(5)P or PtdIns but peak SR Ca²⁺ release was depressed by ~30% and 50% in fibres injected with PtdIns(3,5)P ₂ and PtdIns(3)P, respectively, with no concurrent alteration in the membrane current signals associated with the DHPR function as well as in the voltage dependence of Ca²⁺ release inactivation. In permeabilized muscle fibres, the frequency of spontaneous Ca²⁺ release events was depressed in the presence of the three tested phosphorylated forms of PtdInsP with PtdIns(3,5)P ₂ being the most effective, leading to an almost complete disappearance of Ca²⁺ release events. Results support the possibility that pathological accumulation of MTM1 substrates may acutely depress ryanodine receptor-mediated Ca²⁺ release. Overexpression of a mCherry-tagged form of MTM1 in muscle fibres revealed a striated pattern consistent with the triadic area. Ca²⁺ release remained although unaffected by MTM1 overexpression and was also unaffected by the PtdIns-3-kinase inhibitor LY2940002, suggesting that the 3-phosphorylated PtdIns lipids active on voltage-activated Ca²⁺ release are inherently maintained at a low level, inefficient on Ca²⁺ release in normal conditions.