Contribution of Hfq to gene regulation and virulence in Histophilus somni.

Histophilus somni is one of the predominant bacterial pathogens responsible for bovine respiratory and systemic diseases in cattle. Despite the identification of numerous H. somni virulence factors, little is known about the regulation of such factors. The post-transcriptional regulatory protein Hfq may play a crucial role in regulation of components that affect bacterial virulence. The contribution of Hfq to H. somni phenotype and virulence was investigated following creation of an hfq deletion mutant of H. somni strain 2336 (designated H. somni 2336Δhfq). A comparative analysis of the mutant to the wild-type strain was carried out by examining protein and carbohydrate phenotype, RNA sequence, intracellular survival in bovine monocytes, serum susceptibility, and virulence studies in mouse and calf models. H. somni 2336Δhfq exhibited a truncated lipooligosaccharide (LOS) structure, with loss of sialylation. The mutant demonstrated increased susceptibility to intracellular and serum-mediated killing compared to the wild-type strain. Transcriptomic analysis displayed significant differential expression of 832 upregulated genes and 809 downregulated genes in H. somni 2336Δhfq compared to H. somni strain 2336, including significant downregulation of lsgB and licA, which contribute to LOS oligosaccharide synthesis and sialylation. A substantial number of differentially expressed genes were associated with polysaccharide synthesis and other proteins that could influence virulence. The H. somni 2336Δhfq mutant strain was attenuated in a mouse septicemia model and somewhat attenuated in a calf intrabronchial challenge model. H. somni was recovered less frequently from nasopharyngeal swabs, endotracheal aspirates, and lung tissues of calves challenged with H. somni 2336Δhfq compared to the wild-type strain, and the percentage of abnormal lung tissue in calves challenged with H. somni 2336Δhfq was lower than in calves challenged with the wild-type strain. In conclusion, our results support that Hfq accounts for the regulation of H. somni virulence factors.

Species-agnostic polymeric formulations for inhalable messenger RNA delivery to the lung.

Messenger RNA has now been used to vaccinate millions of people. However, the diversity of pulmonary pathologies, including infections, genetic disorders, asthma and others, reveals the lung as an important organ to directly target for future RNA therapeutics and preventatives. Here we report the screening of 166 polymeric nanoparticle formulations for functional delivery to the lungs, obtained from a combinatorial synthesis approach combined with a low-dead-volume nose-only inhalation system for mice. We identify P76, a poly-ß-amino-thio-ester polymer, that exhibits increased expression over formulations lacking the thiol component, delivery to different animal species with varying RNA cargos and low toxicity. P76 allows for dose sparing when delivering an mRNA-expressed Cas13a-mediated treatment in a SARS-CoV-2 challenge model, resulting in similar efficacy to a 20-fold higher dose of a neutralizing antibody. Overall, the combinatorial synthesis approach allowed for the discovery of promising polymeric formulations for future RNA pharmaceutical development for the lungs.

The Sialyl Lewis X Glycan Receptor Facilitates Infection of Subtype H7 Avian Influenza A Viruses.

Subtype H7 avian influenza A viruses (IAVs) are enzootic in wild aquatic birds and have caused sporadic spillovers into domestic poultry and humans. Here, we determined the distribution of fucosylated α2,3 sialoglycan (i.e., sialyl Lewis X [SLeX]) in chickens and five common dabbling duck species and the association between SLeX and cell/tissue/host tropisms of H7 IAVs. Receptor binding analyses showed that H7 IAVs bind to both α2,3-linked (SA2,3Gal) and α2,6-linked sialic acids (SA2,6Gal), but with a higher preference for SLeX; H7 IAVs replicated more efficiently in SLeX-overexpressed than SLeX-deficient MDCK cells. While chickens and all tested dabbling ducks expressed abundant SA2,3Gal and SA2,6Gal, SLeX was detected in both respiratory and gastrointestinal tissues of chickens and mallard ducks and in only the respiratory tissues of gadwall, green-wing teal, and northern shoveler but not in wood ducks. Viral-tissue binding assays showed that H7 IAVs bind to chicken colon crypt cells that express SLeX but fewer bind to mallard colon crypt cells, which do not express SLeX; H7 IAVs bind efficiently to epithelial cells of all tissues expressing SA2,3Gal. High viral replication was identified in both chickens and mallards infected with an H7 virus, regardless of SLeX expression, and viruses were detected in all cells to the same degree as viruses detected in the viral-tissue binding assays. In summary, this study suggests that SLeX facilitates infection of H7 viruses, but other types of SA2,3Gal glycan receptors shape the tissue/host tropisms of H7 IAVs. IMPORTANCE In addition to causing outbreaks in domestic poultry, subtype H7 IAVs can cause sporadic spillover infections in lower mammals and humans. In this study, we showed that SLeX expression varies among wild dabbling ducks. Although it facilitated virus binding and affected infection of H7 IAV in cells, SLeX expression is not the only determinant of viral replication at either the tissue or host level. This study suggested that access to heterologous SA2,3Gal glycan receptors, including fucosylated α2,3-linked sialoglycans, shape tissue and host tropism of H7 IAVs in aquatic wild birds.

Mitochondrial cardiolipin is required for Nlrp3 inflammasome activation.

Nlrp3 inflammasome activation occurs in response to numerous agonists but the specific mechanism by which this takes place remains unclear. All previously evaluated activators of the Nlrp3 inflammasome induce the generation of mitochondrial reactive oxygen species (ROS), suggesting a model in which ROS is a required upstream mediator of Nlrp3 inflammasome activation. Here we have identified the oxazolidinone antibiotic linezolid as a Nlrp3 agonist that activates the Nlrp3 inflammasome independently of ROS. The pathways for ROS-dependent and ROS-independent Nlrp3 activation converged upon mitochondrial dysfunction and specifically the mitochondrial lipid cardiolipin. Cardiolipin bound to Nlrp3 directly and interference with cardiolipin synthesis specifically inhibited Nlrp3 inflammasome activation. Together these data suggest that mitochondria play a critical role in the activation of the Nlrp3 inflammasome through the direct binding of Nlrp3 to cardiolipin.

Tissue Tropisms of Avian Influenza A Viruses Affect Their Spillovers from Wild Birds to Pigs.

Wild aquatic birds maintain a large, genetically diverse pool of influenza A viruses (IAVs), which can be transmitted to lower mammals and, ultimately, humans. Through phenotypic analyses of viral replication efficiency, only a small set of avian IAVs were found to replicate well in epithelial cells of the swine upper respiratory tract, and these viruses were shown to infect and cause virus shedding in pigs. Such a phenotypic trait of the viral replication efficiency appears to emerge randomly and is distributed among IAVs across multiple avian species and geographic and temporal orders. It is not determined by receptor binding preference but is determined by other markers across genomic segments, such as those in the ribonucleoprotein complex. This study demonstrates that phenotypic variants of viral replication efficiency exist among avian IAVs but that only a few of these may result in viral shedding in pigs upon infection, providing opportunities for these viruses to become adapted to pigs, thus posing a higher potential risk for creating novel variants or detrimental reassortants within pig populations.IMPORTANCE Swine serve as a mixing vessel for generating pandemic strains of human influenza virus. All hemagglutinin subtypes of IAVs can infect swine; however, only sporadic cases of infection with avian IAVs are reported in domestic swine. The molecular mechanisms affecting the ability of avian IAVs to infect swine are still not fully understood. From the findings of phenotypic analyses, this study suggests that the tissue tropisms (i.e., in swine upper respiratory tracts) of avian IAVs affect their spillovers from wild birds to pigs. It was found that this phenotype is determined not by receptor binding preference but is determined by other markers across genomic segments, such as those in the ribonucleoprotein complex. In addition, our results show that such a phenotypic trait was sporadically and randomly distributed among IAVs across multiple avian species and geographic and temporal orders. This study suggests an efficient way for assessment of the risk posed by avian IAVs, such as in evaluating their potentials to be transmitted from birds to pigs.

Aerosol Delivery of Synthetic mRNA to Vaginal Mucosa Leads to Durable Expression of Broadly Neutralizing Antibodies against HIV.

There is a clear need for low-cost, self-applied, long-lasting approaches to prevent human immunodeficiency virus (HIV) infection in both men and women, even with the advent of pre-exposure prophylaxis (PrEP). Broadly neutralizing antibodies represent an option to improve HIV prophylaxis, but intravenous delivery, cold-chain stability requirements, low cervicovaginal concentrations, and cost may preclude their use. Here, we present an approach to express the anti-GP120 broadly neutralizing antibody PGT121 in the primary site of inoculation, the female reproductive tract, using synthetic mRNA. Expression is achieved through aerosol delivery of unformulated mRNA in water. We demonstrated high levels of antibody expression for over 28 days with a single mRNA administration in the reproductive tract of sheep. In rhesus macaques, neutralizing antibody titers in secretions developed within 4 h and simian-HIV (SHIV) infection of ex vivo explants was prevented. Persistence of PGT121 in vaginal secretions and epithelium was achieved through the incorporation of a glycosylphosphatidylinositol (GPI) anchor into the heavy chain of the antibody. Overall, we present a new paradigm to deliver neutralizing antibodies to the female reproductive tract for the prevention of HIV infections.

Virulence and live vaccine potential of Edwardsiella piscicida phoP and phoQ mutants in catfish against edwardsiellosis.

Edwardsiella piscicida is a Gram-negative facultative intracellular bacterium causing edwardsiellosis in catfish, the largest aquaculture industry in the United States. A safe and effective vaccine is an urgent need to avoid economic losses associated with E. piscicida outbreaks. PhoP/PhoQ is a two-component signal transduction system (TCS) that plays an important role in bacterial pathogenesis through sense and response to environmental and host stress signals. This study aimed to explore the contribution of PhoQ/PhoP in E. piscicida virulence and develop live attenuated vaccines against E. piscicida infection in channel catfish (Ictalurus punctatus) and hybrid catfish (channel catfish ♀ × blue catfish (I. furcatus) ♂). In the current study, two in-frame deletion mutants were constructed by deleting phoP (ETAC_09785) and phoQ (ETAC_09790) genes in E. piscicida strain C07-087, and the virulence and protection efficacy of the constructed strains were evaluated in catfish following intraperitoneal injection. Both EpΔphoP and EpΔphoQ strains had a delayed adaptation to oxidative stress (0.2% H2 O2 ) compared to E. piscicida wild type. The EpΔphoP and EpΔphoQ mutants produced significantly less biofilm compared to wild-type E. piscicida. Notably, EpΔphoP and EpΔphoQ mutants were significantly attenuated in channel catfish compared with wild-type E. piscicida (6.63% and 4.17% versus 49.16% mortalities), and channel catfish vaccinated with EpΔphoP and EpΔphoQ were significantly protected (95.65% and 97.92% survival) against E. piscicida infection at 21 days post-vaccination. In hybrid catfish, EpΔphoP was significantly more attenuated than EpΔphoQ, but EpΔphoQ provided significantly better protection than EpΔphoP. EpΔphoP and EpΔphoQ strains both induced specific antibodies in channel catfish against E. piscicida at 14 and 21 days post-vaccination. This result indicated that EpΔphoP and EpΔphoQ mutants were safe and protective in channel catfish fingerlings, while EpΔphoP was safe in hybrid catfish. Our findings show that PhoP and PhoQ are required for adaptation to oxidative stress and biofilm formation and may help E. piscicida face tough environmental challenges; thus, functional PhoP and PhoQ are critical for a successful infection.

Contributions of a LysR Transcriptional Regulator to Listeria monocytogenes Virulence and Identification of Its Regulons.

The capacity of Listeria monocytogenes to adapt to environmental changes is facilitated by a large number of regulatory proteins encoded by its genome. Among these proteins are the uncharacterized LysR-type transcriptional regulators (LTTRs). LTTRs can work as positive and/or negative transcription regulators at both local and global genetic levels. Previously, our group determined by comparative genome analysis that one member of the LTTRs (NCBI accession no. WP_003734782) was present in pathogenic strains but absent from nonpathogenic strains. The goal of the present study was to assess the importance of this transcription factor in the virulence of L. monocytogenes strain F2365 and to identify its regulons. An L. monocytogenes strain lacking lysR (the F2365ΔlysR strain) displayed significant reductions in cell invasion of and adhesion to Caco-2 cells. In plaque assays, the deletion of lysR resulted in a 42.86% decrease in plaque number and a 13.48% decrease in average plaque size. Furthermore, the deletion of lysR also attenuated the virulence of L. monocytogenes in mice following oral and intraperitoneal inoculation. The analysis of transcriptomics revealed that the transcript levels of 139 genes were upregulated, while 113 genes were downregulated in the F2365ΔlysR strain compared to levels in the wild-type bacteria. lysR-repressed genes included ABC transporters, important for starch and sucrose metabolism as well as glycerolipid metabolism, flagellar assembly, quorum sensing, and glycolysis/gluconeogenesis. Conversely, lysR activated the expression of genes related to fructose and mannose metabolism, cationic antimicrobial peptide (CAMP) resistance, and beta-lactam resistance. These data suggested that lysR contributed to L. monocytogenes virulence by broad impact on multiple pathways of gene expression.IMPORTANCEListeria monocytogenes is the causative agent of listeriosis, an infectious and fatal disease of animals and humans. In this study, we have shown that lysR contributes to Listeria pathogenesis and replication in cell lines. We also highlight the importance of lysR in regulating the transcription of genes involved in different pathways that might be essential for the growth and persistence of L. monocytogenes in the host or under nutrient limitation. Better understanding L. monocytogenes pathogenesis and the role of various virulence factors is necessary for further development of prevention and control strategies.

Aerosol Transmission of Gull-Origin Iceland Subtype H10N7 Influenza A Virus in Ferrets.

Subtype H10 influenza A viruses (IAVs) have been recovered from domestic poultry and various aquatic bird species, and sporadic transmission of these IAVs from avian species to mammals (i.e., human, seal, and mink) are well documented. In 2015, we isolated four H10N7 viruses from gulls in Iceland. Genomic analyses showed four gene segments in the viruses were genetically associated with H10 IAVs that caused influenza outbreaks and deaths among European seals in 2014. Antigenic characterization suggested minimal antigenic variation among these H10N7 isolates and other archived H10 viruses recovered from human, seal, mink, and various avian species in Asia, Europe, and North America. Glycan binding preference analyses suggested that, similar to other avian-origin H10 IAVs, these gull-origin H10N7 IAVs bound to both avian-like alpha 2,3-linked sialic acids and human-like alpha 2,6-linked sialic acids. However, when the gull-origin viruses were compared with another Eurasian avian-origin H10N8 IAV, which caused human infections, the gull-origin virus showed significantly higher binding affinity to human-like glycan receptors. Results from a ferret experiment demonstrated that a gull-origin H10N7 IAV replicated well in turbinate, trachea, and lung, but replication was most efficient in turbinate and trachea. This gull-origin H10N7 virus can be transmitted between ferrets through the direct contact and aerosol routes, without prior adaptation. Gulls share their habitat with other birds and mammals and have frequent contact with humans; therefore, gull-origin H10N7 IAVs could pose a risk to public health. Surveillance and monitoring of these IAVs at the wild bird-human interface should be continued.IMPORTANCE Subtype H10 avian influenza A viruses (IAVs) have caused sporadic human infections and enzootic outbreaks among seals. In the fall of 2015, H10N7 viruses were recovered from gulls in Iceland, and genomic analyses showed that the viruses were genetically related with IAVs that caused outbreaks among seals in Europe a year earlier. These gull-origin viruses showed high binding affinity to human-like glycan receptors. Transmission studies in ferrets demonstrated that the gull-origin IAV could infect ferrets, and that the virus could be transmitted between ferrets through direct contact and aerosol droplets. This study demonstrated that avian H10 IAV can infect mammals and be transmitted among them without adaptation. Thus, avian H10 IAV is a candidate for influenza pandemic preparedness and should be monitored in wildlife and at the animal-human interface.

Tissue tropisms opt for transmissible reassortants during avian and swine influenza A virus co-infection in swine.

Genetic reassortment between influenza A viruses (IAVs) facilitate emergence of pandemic strains, and swine are proposed as a "mixing vessel" for generating reassortants of avian and mammalian IAVs that could be of risk to mammals, including humans. However, how a transmissible reassortant emerges in swine are not well understood. Genomic analyses of 571 isolates recovered from nasal wash samples and respiratory tract tissues of a group of co-housed pigs (influenza-seronegative, avian H1N1 IAV-infected, and swine H3N2 IAV-infected pigs) identified 30 distinct genotypes of reassortants. Viruses recovered from lower respiratory tract tissues had the largest genomic diversity, and those recovered from turbinates and nasal wash fluids had the least. Reassortants from lower respiratory tracts had the largest variations in growth kinetics in respiratory tract epithelial cells, and the cold temperature in swine nasal cells seemed to select the type of reassortant viruses shed by the pigs. One reassortant in nasal wash samples was consistently identified in upper, middle, and lower respiratory tract tissues, and it was confirmed to be transmitted efficiently between pigs. Study findings suggest that, during mixed infections of avian and swine IAVs, genetic reassortments are likely to occur in the lower respiratory track, and tissue tropism is an important factor selecting for a transmissible reassortant.

Mutation W222L at the Receptor Binding Site of Hemagglutinin Could Facilitate Viral Adaption from Equine Influenza A(H3N8) Virus to Dogs.

An outbreak of respiratory disease caused by the equine-origin influenza A(H3N8) virus was first detected in dogs in 2004 and since then has been enzootic among dogs. Currently, the molecular mechanisms underlying host adaption of this virus from horses to dogs is unknown. Here, we have applied quantitative binding, growth kinetics, and immunofluorescence analyses to elucidate these mechanisms. Our findings suggest that a substitution of W222L in the hemagglutinin of the equine-origin A(H3N8) virus facilitated its host adaption to dogs. This mutation increased binding avidity of the virus specifically to receptor glycans with N-glycolylneuraminic acid (Neu5Gc) and sialyl Lewis X (SLeX) motifs. We have demonstrated these motifs are abundantly located in the submucosal glands of dog trachea. Our findings also suggest that in addition to the type of glycosidic linkage (e.g., α2,3-linkage or α2,6-linkage), the type of sialic acid (Neu5Gc or 5-N-acetyl neuraminic acid) and the glycan substructure (e.g., SLeX) also play an important role in host tropism of influenza A viruses.IMPORTANCE Influenza A viruses (IAVs) cause a significant burden on human and animal health, and mechanisms for interspecies transmission of IAVs are far from being understood. Findings from this study suggest that an equine-origin A(H3N8) IAV with mutation W222L at its hemagglutinin increased binding to canine-specific receptors with sialyl Lewis X and Neu5Gc motifs and, thereby, may have facilitated viral adaption from horses to dogs. These findings suggest that in addition to the glycosidic linkage (e.g., α2,3-linked and α2,6-linked), the substructure in the receptor saccharides (e.g., sialyl Lewis X and Neu5Gc) could present an interspecies transmission barrier for IAVs and drive viral mutations to overcome such barriers.

Influenza D Virus Infection in Feral Swine Populations, United States.

Influenza D virus (IDV) has been identified in domestic cattle, swine, camelid, and small ruminant populations across North America, Europe, Asia, South America, and Africa. Our study investigated seroprevalence and transmissibility of IDV in feral swine. During 2012-2013, we evaluated feral swine populations in 4 US states; of 256 swine tested, 57 (19.1%) were IDV seropositive. Among 96 archived influenza A virus-seropositive feral swine samples collected from 16 US states during 2010-2013, 41 (42.7%) were IDV seropositive. Infection studies demonstrated that IDV-inoculated feral swine shed virus 3-5 days postinoculation and seroconverted at 21 days postinoculation; 50% of in-contact naive feral swine shed virus, seroconverted, or both. Immunohistochemical staining showed viral antigen within epithelial cells of the respiratory tract, including trachea, soft palate, and lungs. Our findings suggest that feral swine might serve an important role in the ecology of IDV.

TRAF2 exerts opposing effects on basal and TNFα-induced activation of the classic IKK complex in hematopoietic cells in mice.

The role of TRAF2 and TRAF5 in TNFα-induced NF-κB activation has become complicated owing to the accumulation of conflicting data. Here, we report that 7-day-old TRAF2-knockout (KO) and TRAF2 TRAF5 double KO (TRAF2/5-DKO) mice exhibit enhanced canonical IκB kinase (IKK) and caspase-8 activation in spleen and liver, and that subsequent knockout of TNFα suppresses the basal activity of caspase-8, but not of IKK. In primary TRAF2 KO and TRAF2/5-DKO cells, TNFα-induced immediate IKK activation is impaired, whereas delayed IKK activation occurs normally; as such, owing to elevated basal and TNFα-induced delayed IKK activation, TNFα stimulation leads to significantly increased induction of a subset of NF-κB-dependent genes in these cells. In line with this, both TRAF2 KO and TRAF2/5-DKO mice succumb to a sublethal dose of TNFα owing to increased expression of NF-κB target genes, diarrhea and bradypnea. Notably, depletion of IAP1 and IAP2 (also known as BIRC2 and BIRC3, respectively) also results in elevated basal IKK activation that is independent of autocrine TNFα production and that impairs TNFα-induced immediate IKK activation. These data reveal that TRAF2, IAP1 and IAP2, but not TRAF5, cooperatively regulate basal and TNFα-induced immediate IKK activation.

Sirt3-mediated deacetylation of evolutionarily conserved lysine 122 regulates MnSOD activity in response to stress.

Genetic deletion of the mitochondrial deacetylase sirtuin-3 (Sirt3) results in increased mitochondrial superoxide, a tumor-permissive environment, and mammary tumor development. MnSOD contains a nutrient- and ionizing radiation (IR)-dependent reversible acetyl-lysine that is hyperacetylated in Sirt3⁻/⁻ livers at 3 months of age. Livers of Sirt3⁻/⁻ mice exhibit decreased MnSOD activity, but not immunoreactive protein, relative to wild-type livers. Reintroduction of wild-type but not deacetylation null Sirt3 into Sirt3⁻/⁻ MEFs deacetylated lysine and restored MnSOD activity. Site-directed mutagenesis of MnSOD lysine 122 to an arginine, mimicking deacetylation (lenti-MnSOD(K122-R)), increased MnSOD activity when expressed in MnSOD⁻/⁻ MEFs, suggesting acetylation directly regulates function. Furthermore, infection of Sirt3⁻/⁻ MEFs with lenti-MnSOD(K122-R) inhibited in vitro immortalization by an oncogene (Ras), inhibited IR-induced genomic instability, and decreased mitochondrial superoxide. Finally, IR was unable to induce MnSOD deacetylation or activity in Sirt3⁻/⁻ livers, and these irradiated livers displayed significant IR-induced cell damage and microvacuolization in their hepatocytes.

Pathogenesis of Influenza D Virus in Cattle.

UNLABELLED: Cattle have been proposed as the natural reservoir of a novel member of the virus family Orthomyxoviridae, which has been tentatively classified as influenza D virus (IDV). Although isolated from sick animals, it is unclear whether IDV causes any clinical disease in cattle. To address this aspect of Koch's postulates, three dairy calves (treatment animals) held in individual pens were inoculated intranasally with IDV strain D/bovine/Mississippi/C00046N/2014. At 1 day postinoculation, a seronegative calf (contact animal) was added to each of the treatment animal pens. The cattle in both treatment and contact groups seroconverted, and virus was detected in their respiratory tracts. Histologically, there was a significant increase in neutrophil tracking in tracheal epithelia of the treatment calves compared to control animals. While infected and contact animals demonstrated various symptoms of respiratory tract infection, they were mild, and the calves in the treatment group did not differ from the controls in terms of heart rate, respiratory rate, or rectal temperature. To mimic zoonotic transmission, two ferrets were exposed to a plastic toy fomite soaked with infected nasal discharge from the treatment calves. These ferrets did not shed the virus or seroconvert. In summary, this study demonstrates that IDV causes a mild respiratory disease upon experimental infection of cattle and can be transmitted effectively among cattle by in-pen contact, but not from cattle to ferrets through fomite exposure. These findings support the hypothesis that cattle are a natural reservoir for the virus. IMPORTANCE: A novel influenza virus, tentatively classified as influenza D virus (IDV), was identified in swine, cattle, sheep, and goats. Among these hosts, cattle have been proposed as the natural reservoir. In this study, we show that cattle experimentally infected with IDV can shed virus and transmit it to other cattle through direct contact, but not to ferrets through fomite routes. IDV caused minor clinical signs in the infected cattle, fulfilling another of Koch's postulates for this novel agent, although other objective clinical endpoints were not different from those of control animals. Although the disease observed was mild, IDV induced neutrophil tracking and epithelial attenuation in cattle trachea, which could facilitate coinfection with other pathogens, and in doing so, predispose animals to bovine respiratory disease.

Inflammasome-independent IL-1ß mediates autoinflammatory disease in Pstpip2-deficient mice.

Chronic recurrent multifocal osteomyelitis (CRMO) is a human autoinflammatory disorder that primarily affects bone. Missense mutation (L98P) of proline-serine-threonine phosphatase-interacting protein 2 (Pstpip2) in mice leads to a disease that is phenotypically similar to CRMO called chronic multifocal osteomyelitis (cmo). Here we show that deficiency of IL-1RI in cmo mice resulted in a significant reduction in the time to onset of disease as well as the degree of bone pathology. Additionally, the proinflammatory cytokine IL-1ß, but not IL-1α, played a critical role in the pathology observed in cmo mice. In contrast, disease in cmo mice was found to be independent of the nucleotide-binding domain, leucine-rich repeat-containing family, pyrin domain-containing 3 (NLRP3) inflammasome as well as caspase-1. Neutrophils, but not bone marrow-derived macrophages, from cmo mice secreted increased IL-1ß in response to ATP, silica, and Pseudomonas aeruginosa compared with neutrophils from WT mice. This aberrant neutrophil response was sensitive to inhibition by serine protease inhibitors. These results demonstrate an inflammasome-independent role for IL-1ß in disease progression of cmo and implicate neutrophils and neutrophil serine proteases in disease pathogenesis. These data provide a rationale for directly targeting IL-1RI or IL-1ß as a therapeutic strategy in CRMO.

Functional validation of a human CAPN5 exome variant by lentiviral transduction into mouse retina.

Exome sequencing indicated that the gene encoding the calpain-5 protease, CAPN5, is the likely cause of retinal degeneration and autoimmune uveitis in human patients with autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV, OMIM #193235). To explore the mechanism of ADNIV, a human CAPN5 disease allele was expressed in mouse retinas with a lentiviral vector created to express either the wild-type human (h) CAPN5 or the ADNIV mutant hCAPN5-R243L allele under a rhodopsin promoter with tandem green fluorescent protein (GFP) expression. Vectors were injected into the subretinal space of perinatal mice. Mouse phenotypes were analyzed using electroretinography, histology and inflammatory gene expression profiling. Mouse calpain-5 showed high homology to its human ortholog with >98% sequence identity that includes the ADNIV mutant residue. Calpain-5 protein was expressed in the inner and outer segments of the photoreceptors and in the outer plexiform layer. Expression of the hCAPN5-R243L allele caused loss of the electroretinogram b-wave, photoreceptor degeneration and induction of immune cell infiltration and inflammatory genes in the retina, recapitulating major features of the ADNIV phenotype. Intraocular neovascularization and fibrosis were not observed during the study period. Our study shows that expression of the hCAPN5-R243L disease allele elicits an ADNIV-like disease in mice. It further suggests that ADNIV is due to CAPN5 gain-of-function rather than haploinsufficiency, and retinal expression may be sufficient to generate an autoimmune response. Genetic models of ADNIV in the mouse can be used to explore protease mechanisms in retinal degeneration and inflammation as well as preclinical therapeutic testing.

Immune complexes inhibit IL-1 secretion and inflammasome activation.

IgG immune complexes have been shown to modify immune responses driven by APCs in either a pro- or anti-inflammatory direction depending upon the context of stimulation. However, the ability of immune complexes to modulate the inflammasome-dependent innate immune response is unknown. In this study, we show that IgG immune complexes suppress IL-1α and IL-1ß secretion through inhibition of inflammasome activation. The mechanism by which this inhibition occurs is via immune complex ligation of activating FcγRs, resulting in prevention of both activation and assembly of the inflammasome complex in response to nucleotide-binding domain leucine-rich repeat (NLR) P3, NLRC4, or AIM2 agonists. In vivo, administration of Ag in the form of an immune complex during priming of the immune response inhibited resultant adaptive immune responses in an NLRP3-dependent model of allergic airway disease. Our data reveal an unexpected mechanism regulating CD4(+) T cell differentiation, by which immune complexes suppress inflammasome activation and the generation of IL-1α and IL-1ß from APCs, which are critical for the Ag-driven differentiation of CD4(+) T cells.

Strain- and time-dependent alterations in hepatic iron metabolism in a murine model of nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis is a common liver disease that is often accompanied by dysregulated iron metabolism. The aim of the study was to test the hypothesis that aberrant iron metabolism in nonalcoholic steatohepatitis is modulated by genetic susceptibility to inflammation and oxidative stress. Hepatic histology and iron content were assessed in 3 inbred strains of mice (C57BL/6, BALB/c, and C3H/HeJ) fed an atherogenic diet (AD). Hepatic expression of genes relevant to iron metabolism, inflammation, and oxidative stress were quantitated by real-time reverse transcription-polymerase chain reaction. At 6 weeks on the AD, histologic injury and induction of inflammatory and oxidative stress-associated gene expression were most pronounced in C57BL/6. At 18 weeks on the AD, these parameters were similar in C57BL/6 and BALB/c. Atherogenic diet-fed C3H/HeJ showed milder responses at both time points. The AD was associated with decreased hepatic iron concentrations in all strains at 6 and 18 weeks. The decrease in hepatic iron concentrations did not correlate with changes in hepcidin expression and was not associated with altered expression of iron transporters. These findings are similar to those observed in models of obesity-induced steatosis and indicate that hepatic steatosis can be associated with depletion of iron stores that is not explained by upregulation of hepcidin expression by inflammation. SIGNIFICANCE OF THE STUDY: Nonalcoholic steatohepatitis (NASH) is a common liver disease that often accompanies the metabolic syndrome. The latter condition has been linked to iron deficiency and diminished intestinal iron absorption, likely the result of hepcidin upregulation by chronic inflammation. Paradoxically, some NASH patients accumulate excess hepatic iron, which may increase fibrosis and cancer risk. Iron accumulation has been attributed to suppression of hepcidin by oxidative stress. The objective of this study was to investigate the contributions of inflammation and oxidative stress to altered hepatic iron metabolism in a murine model of NASH using inbred strains of mice with differing susceptibilities to injury.

Animal models of gastrointestinal and liver diseases. Animal models of cystic fibrosis: gastrointestinal, pancreatic, and hepatobiliary disease and pathophysiology.

Multiple organ systems, including the gastrointestinal tract, pancreas, and hepatobiliary systems, are affected by cystic fibrosis (CF). Many of these changes begin early in life and are difficult to study in young CF patients. Recent development of novel CF animal models has expanded opportunities in the field to better understand CF pathogenesis and evaluate traditional and innovative therapeutics. In this review, we discuss manifestations of CF disease in gastrointestinal, pancreatic, and hepatobiliary systems of humans and animal models. We also compare the similarities and limitations of animal models and discuss future directions for modeling CF.