HLA-II-Dependent Neuroimmune Changes in Group A Streptococcal Necrotizing Fasciitis

Streptococcus pyogenes (Group A Streptococcus, GAS) bacteria cause a spectrum of human diseases ranging from self-limiting pharyngitis and mild, uncomplicated skin infections (impetigo, erysipelas, and cellulitis) to highly morbid and rapidly invasive, life-threatening infections such as streptococcal toxic shock syndrome and necrotizing fasciitis (NF). HLA class II allelic polymorphisms are linked with differential outcomes and severity of GAS infections. The dysregulated immune response and peripheral cytokine storm elicited due to invasive GAS infections increase the risk for toxic shock and multiple organ failure in genetically susceptible individuals. We hypothesized that, while the host immune mediators regulate the immune responses against peripheral GAS infections, these interactions may simultaneously trigger neuropathology and, in some cases, induce persistent alterations in the glial phenotypes. Here, we studied the consequences of peripheral GAS skin infection on the brain in an HLA-II transgenic mouse model of GAS NF with and without treatment with an antibiotic, clindamycin (CLN). Mice expressing the human HLA-II DR3 (DR3) or the HLA-II DR4 (DR4) allele were divided into three groups: (i) uninfected controls, (ii) subcutaneously infected with a clinical GAS strain isolated from a patient with GAS NF, and (iii) GAS-infected with CLN treatment (10 mg/kg/5 days, intraperitoneal). The groups were monitored for 15 days post-infection. Skin GAS burden and lesion area, splenic and hippocampal mRNA levels of inflammatory markers, and immunohistochemical changes in hippocampal GFAP and Iba-1 immunoreactivity were assessed. Skin GAS burden and hippocampal mRNA levels of the inflammatory markers S100A8/A9, IL-1β, IL-33, inflammasome-related caspase-1 (Casp1), and NLRP6 were elevated in infected DR3 but not DR4 mice. The levels of these markers were significantly reduced following CLN treatment in DR3 mice. Although GAS was not detectable in the brain, astrocyte (GFAP) and microglia (Iba-1) activation were evident from increased GFAP and Iba-1 mRNA levels in DR3 and DR4 mice. However, CLN treatment significantly reduced GFAP mRNA levels in DR3 mice, not DR4 mice. Our data suggest a skin–brain axis during GAS NF, demonstrating that peripherally induced pathological conditions regulate neuroimmune changes and gliotic events in the brai

Distress call-induced gene expression in the brain of the Indian short-nosed fruit bat, Cynopterus sphinx

Individuals in distress emit audible vocalizations to either warn or inform conspecifics. The Indian short-nosed fruit bat, Cynopterus sphinx, emits distress calls soon after becoming entangled in mist nets, which appear to attract conspecifics. Phase I of these distress calls is longer and louder, and includes a secondary peak, compared to phase II. Activity-dependent expression of egr-1 was examined in free-ranging C. sphinx following the emissions and responses to a distress call. We found that the level of expression of egr-1 was higher in bats that emitted a distress call, in adults that responded, and in pups than in silent bats. Up-regulated cDNA was amplified to identify the target gene (TOE1) of the protein Egr-1. The observed expression pattern Toe1 was similar to that of egr-1. These findings suggest that the neuronal activity related to recognition of a distress call and an auditory feedback mechanism induces the expression of Egr-1. Co-expression of egr-1 with Toe1 may play a role in initial triggering of the genetic mechanism that could be involved in the consolidation or stabilization of distress call memories

Identification of a functionally distinct truncated BDNF mRNA splice variant and protein in Trachemys scripta elegans.

Brain-derived neurotrophic factor (BDNF) has a diverse functional role and complex pattern of gene expression. Alternative splicing of mRNA transcripts leads to further diversity of mRNAs and protein isoforms. Here, we describe the regulation of BDNF mRNA transcripts in an in vitro model of eyeblink classical conditioning and a unique transcript that forms a functionally distinct truncated BDNF protein isoform. Nine different mRNA transcripts from the BDNF gene of the pond turtle Trachemys scripta elegans (tBDNF) are selectively regulated during classical conditioning: exon I mRNA transcripts show no change, exon II transcripts are downregulated, while exon III transcripts are upregulated. One unique transcript that codes from exon II, tBDNF2a, contains a 40 base pair deletion in the protein coding exon that generates a truncated tBDNF protein. The truncated transcript and protein are expressed in the naïve untrained state and are fully repressed during conditioning when full-length mature tBDNF is expressed, thereby having an alternate pattern of expression in conditioning. Truncated BDNF is not restricted to turtles as a truncated mRNA splice variant has been described for the human BDNF gene. Further studies are required to determine the ubiquity of truncated BDNF alternative splice variants across species and the mechanisms of regulation and function of this newly recognized BDNF protein

Role of olfactory bulb serotonin in olfactory learning in the greater short-nosed fruit bat, Cynopterus sphinx (Chiroptera: Pteropodidae)

The role of olfactory bulb (OB) serotonin [5-hydroxytryptamine (5-HT)] in olfactory learning and memory was tested in the greater short-nosed fruit bat, Cynopterus sphinx (family Pteropodidae). Graded concentrations (25, 40, and 60 μg) of 5,7-dihydroxytryptamine (5,7-DHT) or saline were injected into the OB of bats one day before training to the novel odor. In a behavioral test, 5,7-DHT (60 μg) injected bats made significantly fewer feeding attempts and bouts when compared to saline-injected bats during learning and in the memory test. Subsequent biochemical analysis showed that 5-HT level was effectively depleted in the OB of 5,7-DHT injected bats. To test odor-induced 5-HT mediated changes in 5-HT receptors and second messenger cascade in the OB, we examined the expression of 5-HT receptors and mitogen-activated protein kinase (MAPK)/Erk cascade after training to the novel odor. We found that odor stimulation up-regulated the expression of 5-HT1A receptor, Erk1 and Creb1 mRNA, and phosphorylation of ERK1 and CREB1. Odor stimulation failed to induce expression in 5-HT-depleted bats, which is similar to control bats and significantly low compared to saline-treated bats. Together these data revealed that the level of 5-HT in the OB may regulate olfactory learning and memory in C. sphinx through Erk and CREB

<i>BDNF</i> gene structure in turtle and alternatively spliced mRNA transcripts.

<p>Schematic illustration of the <i>BDNF</i> gene structure in turtle and the nine alternatively spliced mRNA transcripts identified here. The 5′ exons I, II, and III are spliced to a common protein coding exon IV that encodes the preproBDNF protein to form three classes of transcripts designated <i>tBDNF1-3</i>. Exon I contains a translation initiation site that codes for 8 additional amino acids at the N-terminal end of the BDNF protein and exons II and III are non-coding exons. Due to alternative splicing of three polyadenylation sites, the transcripts may have short, intermediate or long 3′ UTRs. One unique transcript, <i>tBDNF2a</i>, has a deletion within the coding exon (asterisk) that results in a truncated <i>tBDNF</i> mRNA transcript.</p

Distress call-induced gene expression in the brain of the Indian short-nosed fruit bat, Cynopterus sphinx

Individuals in distress emit audible vocalizations to either warn or inform conspecifics. The Indian short-nosed fruit bat, Cynopterus sphinx, emits distress calls soon after becoming entangled in mist nets, which appear to attract conspecifics. Phase I of these distress calls is longer and louder, and includes a secondary peak, compared to phase II. Activity-dependent expression of egr-1 was examined in free-ranging C. sphinx following the emissions and responses to a distress call. We found that the level of expression of egr-1 was higher in bats that emitted a distress call, in adults that responded, and in pups than in silent bats. Up-regulated cDNA was amplified to identify the target gene (TOE1) of the protein Egr-1. The observed expression pattern Toe1 was similar to that of egr-1. These findings suggest that the neuronal activity related to recognition of a distress call and an auditory feedback mechanism induces the expression of Egr-1. Co-expression of egr-1 with Toe1 may play a role in initial triggering of the genetic mechanism that could be involved in the consolidation or stabilization of distress call memories

Expression of <i>tBDNF</i> mRNA transcripts is selectively regulated during conditioning.

<p>Agarose gels of the PCR products for each of the nine <i>tBDNF</i> mRNA transcripts showing their pattern of expression during the different training procedures examined: naïve (N), pseudoconditioned for one session (Ps1), and conditioned for one session (C1, or after 25 minutes). PCR products for b-actin mRNA transcripts were used for comparison and are shown. Semi-quantitative analysis of mRNA expression of each <i>tBDNF</i> transcript is shown. Transcripts <i>tBDNF2a-c</i> are significantly decreased after conditioning while <i>tBDNF3b</i> is increased by about threefold. The band below 1000 bp for the exon III gel is non-specific.</p

Conditioning induces expression of full-length mature tBDNF protein and suppression of truncated tBDNF.

<p>Western blot analysis using a primary antibody to BDNF showing expression of the full-length 14 kD mature tBDNF protein after one (C1) or two (C2) sessions of conditioning. In the naïve state (N), after pseudoconditioning trials for 15 minutes (Ps₁₅) or one session (Ps1) or early stages of conditioning after 15 minutes (C₁₅), an 11 kD protein band is expressed that represents a truncated tBDNF protein generated by the <i>tBDNF2a</i> transcript. When full-length mature tBDNF is expressed in conditioning, truncated tBDNF is completely suppressed. Expression of either protein isoform is not altered from the naïve state by a generalized increase in activity levels induced by bath application of 50 µM glutamate (Glu2) or 15 mM KCl (KCl2) for the equivalent time period of two sessions. <i>P</i> values are given in the text and are determined relative to the naïve group.</p

Comparison of the amino acid sequence between full-length and truncated human and turtle BDNF.

<p>Amino acid sequence alignment of the coding region for human BDNF 1 and 7 are shown aligned with turtle full-length BDNF and truncated BDNF (labeled as tBDNF2a) to show the two different truncated BDNF sequences. The position of the deleted amino acid sequences for truncated hBDNF7 and tBDNF2a are indicted by the dashes. Differences between human and turtle sequences are highlighted in yellow. The arrow indicates the site of proteolytic cleavage of mature BDNF protein from the proBDNF precursor.</p