Neutrophil-mediated Suppression of Influenza-induced Pathology Requires CD11b/CD18 (MAC-1).

Severe influenza virus infection can lead to life-threatening pathology through immune-mediated tissue damage. In various experimental models, this damage is dependent on T cells. There is conflicting evidence regarding the role of neutrophils in influenza-mediated pathology. Neutrophils are often regarded as cells causing tissue damage, but, in recent years, it has become clear that a subset of human neutrophils is capable of suppressing T cells, which is dependent on macrophage-1 antigen (CD11b/CD18). Therefore, we tested the hypothesis that immune suppression by neutrophils can reduce T cell-mediated pathology after influenza infection. Wild-type (WT) and CD11b-/- mice were infected with A/HK/2/68 (H3N2) influenza virus. Disease severity was monitored by weight loss, leukocyte infiltration, and immunohistochemistry. We demonstrated that CD11b-/- mice suffered increased weight loss compared with WT animals upon infection with influenza virus. This was accompanied by increased pulmonary leukocyte infiltration and lung damage. The exaggerated pathology in CD11b-/- mice was dependent on T cells, as it was reduced by T cell depletion. In addition, pathology in CD11b-/- mice was accompanied by higher numbers of T cells in the lungs early during infection compared with WT mice. Importantly, these differences in pathology were not associated with an increased viral load, suggesting that pathology was immune-mediated rather than caused by virus-induced damage. In contrast to adoptive transfer of CD11b-/- neutrophils, a single adoptive transfer of WT neutrophils partly restored protection against influenza-induced pathology, demonstrating the importance of neutrophil CD11b/CD18. Our data show that neutrophil CD11b/CD18 limits pathology in influenza-induced, T cell-mediated disease.

CD200 receptor controls sex-specific TLR7 responses to viral infection.

Immunological checkpoints, such as the inhibitory CD200 receptor (CD200R), play a dual role in balancing the immune system during microbial infection. On the one hand these inhibitory signals prevent excessive immune mediated pathology but on the other hand they may impair clearance of the pathogen. We studied the influence of the inhibitory CD200-CD200R axis on clearance and pathology in two different virus infection models. We find that lack of CD200R signaling strongly enhances type I interferon (IFN) production and viral clearance and improves the outcome of mouse hepatitis corona virus (MHV) infection, particularly in female mice. MHV clearance is known to be dependent on Toll like receptor 7 (TLR7)-mediated type I IFN production and sex differences in TLR7 responses previously have been reported for humans. We therefore hypothesize that CD200R ligation suppresses TLR7 responses and that release of this inhibition enlarges sex differences in TLR7 signaling. This hypothesis is supported by our findings that in vivo administration of synthetic TLR7 ligand leads to enhanced type I IFN production, particularly in female Cd200(-/-) mice and that CD200R ligation inhibits TLR7 signaling in vitro. In influenza A virus infection we show that viral clearance is determined by sex but not by CD200R signaling. However, absence of CD200R in influenza A virus infection results in enhanced lung neutrophil influx and pathology in females. Thus, CD200-CD200R and sex are host factors that together determine the outcome of viral infection. Our data predict a sex bias in both beneficial and pathological immune responses to virus infection upon therapeutic targeting of CD200-CD200R.

Liver-on-chips for drug discovery and development.

Recent FDA modernization act 2.0 has led to increasing industrial R&D investment in advanced in vitro 3D models such as organoids, spheroids, organ-on-chips, 3D bioprinting, and in silico approaches. Liver-related advanced in vitro models remain the prime area of interest, as liver plays a central role in drug clearance of compounds. Growing evidence indicates the importance of recapitulating the overall liver microenvironment to enhance hepatocyte maturity and culture longevity using liver-on-chips (LoC) in vitro. Hence, pharmaceutical industries have started exploring LoC assays in the two of the most challenging areas: accurate in vitro-in vivo extrapolation (IVIVE) of hepatic drug clearance and drug-induced liver injury. We examine the joint efforts of commercial chip manufacturers and pharmaceutical companies to present an up-to-date overview of the adoption of LoC technology in the drug discovery. Further, several roadblocks are identified to the rapid adoption of LoC assays in the current drug development framework. Finally, we discuss some of the underexplored application areas of LoC models, where conventional 2D hepatic models are deemed unsuitable. These include clearance prediction of metabolically stable compounds, immune-mediated drug-induced liver injury (DILI) predictions, bioavailability prediction with gut-liver systems, hepatic clearance prediction of drugs given during pregnancy, and dose adjustment studies in disease conditions. We conclude the review by discussing the importance of PBPK modeling with LoC, digital twins, and AI/ML integration with LoC.

Reliable means of diagnosis and serovar determination of blood-borne Salmonella strains: quick PCR amplification of unique genomic loci by novel primer sets.

Typhoid fever is becoming an ever increasing threat in the developing countries. We have improved considerably upon the existing PCR-based diagnosis method by designing primers against a region that is unique to Salmonella enterica subsp. enterica serovar Typhi and Salmonella enterica subsp. enterica serovar Paratyphi A, corresponding to the STY0312 gene in S. Typhi and its homolog SPA2476 in S. Paratyphi A. An additional set of primers amplify another region in S. Typhi CT18 and S. Typhi Ty2 corresponding to the region between genes STY0313 to STY0316 but which is absent in S. Paratyphi A. The possibility of a false-negative result arising due to mutation in hypervariable genes has been reduced by targeting a gene unique to typhoidal Salmonella serovars as a diagnostic marker. The amplified region has been tested for genomic stability by amplifying the region from clinical isolates of patients from various geographical locations in India, thereby showing that this region is potentially stable. These set of primers can also differentiate between S. Typhi CT18, S. Typhi Ty2, and S. Paratyphi A, which have stable deletions in this specific locus. The PCR assay designed in this study has a sensitivity of 95% compared to the Widal test which has a sensitivity of only 63%. As observed, in certain cases, the PCR assay was more sensitive than the blood culture test was, as the PCR-based detection could also detect dead bacteria.

lac repressor is an antivirulence factor of Salmonella enterica: its role in the evolution of virulence in Salmonella.

The genus Salmonella includes many pathogens of great medical and veterinary importance. Bacteria belonging to this genus are very closely related to those belonging to the genus Escherichia. lacZYA operon and lacI are present in Escherichia coli, but not in Salmonella enterica. It has been proposed that Salmonella has lost lacZYA operon and lacI during evolution. In this study, we have investigated the physiological and evolutionary significance of the absence of lacI in Salmonella enterica. Using murine model of typhoid fever, we show that the expression of LacI causes a remarkable reduction in the virulence of Salmonella enterica. LacI also suppresses the ability of Salmonella enterica to proliferate inside murine macrophages. Microarray analysis revealed that LacI interferes with the expression of virulence genes of Salmonella pathogenicity island 2. This effect was confirmed by RT-PCR and Western blot analysis. Interestingly, we found that SBG0326 of Salmonella bongori is homologous to lacI of Escherichia coli. Salmonella bongori is the only other species of the genus Salmonella and it lacks the virulence genes of Salmonella pathogenicity island 2. Overall, our results demonstrate that LacI is an antivirulence factor of Salmonella enterica and suggest that absence of lacI has facilitated the acquisition of virulence genes of Salmonella pathogenicity island 2 in Salmonella enterica making it a successful systemic pathogen.

SopB of Salmonella enterica serovar Typhimurium is a potential DNA vaccine candidate in conjugation with live attenuated bacteria.

The immune response against Salmonella is multi-faceted involving both the innate and the adaptive immune system. The characterization of specific Salmonella antigens inducing immune response could critically contribute to the development of epitope based vaccines for Salmonella. We have tried to identify a protective T cell epitope(s) of Salmonella, as cell mediated immunity conferred by CD8+ T cells is the most crucial subset conferring protective immunity against Salmonella. It being a proven fact that secreted proteins are better in inducing cell mediated immunity than cell surface and cytosolic antigens, we have analyzed all the genbank annotated Salmonella pathogenicity island 1 and 2 secreted proteins of Salmonella enterica serovar Typhimurium (S. typhimurium) and S. enterica serovar Typhi (S. typhi). They were subjected to BIMAS and SYFPEITHI analysis to map MHC-I and MHC-II binding epitopes. The huge profile of possible T cell epitopes obtained from the two classes of secreted proteins were tabulated and using a scoring system that considers the binding affinity and promiscuity of binding to more than one allele, SopB and SifB were chosen for experimental confirmation in murine immunization model. The entire SopB and SifB genes were cloned into DNA vaccine vectors and were administered along with live attenuated Salmonella and it was found that SopB vaccination reduced the bacterial burden of organs by about 5-fold on day 4 and day 8 after challenge with virulent Salmonella and proved to be a more efficient vaccination strategy than live attenuated bacteria alone.

Differentially evolved genes of Salmonella pathogenicity islands: insights into the mechanism of host specificity in Salmonella.

BACKGROUND: The species Salmonella enterica (S. enterica) includes many serovars that cause disease in avian and mammalian hosts. These serovars differ greatly in their host range and their degree of host adaptation. The host specificity of S. enterica serovars appears to be a complex phenomenon governed by multiple factors acting at different stages of the infection process, which makes identification of the cause/s of host specificity solely by experimental methods difficult. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we have employed a molecular evolution and phylogenetics based approach to identify genes that might play important roles in conferring host specificity to different serovars of S. enterica. These genes are 'differentially evolved' in different S. enterica serovars. This list of 'differentially evolved' genes includes genes that encode translocon proteins (SipD, SseC and SseD) of both Salmonella pathogenicity islands 1 and 2 encoded type three secretion systems, sptP, which encodes an effector protein that inhibits the mitogen-activated protein kinase pathway of the host cell, and genes which encode effector proteins (SseF and SifA) that are important in placing the Salmonella-containing vacuole in a juxtanuclear position. CONCLUSIONS/SIGNIFICANCE: Analysis of known functions of these 'differentially evolved genes' indicates that the products of these genes directly interact with the host cell and manipulate its functions and thereby confer host specificity, at least in part, to different serovars of S. enterica that are considered in this study.