Distinct host-immune response toward species related intracellular mycobacterial killing : a transcriptomic study

CITATION: Madhvi Abhilasha et al. 2020. Distinct host-immune response toward species related intracellular mycobacterial killing : a transcriptomic study. Virulence, 11(1):170-182, doi:10.1080/21505594.2020.1726561.The original publication is availablle at: https://www.ncbi.nlm.nih.govThe comparison of the host immune response when challenged with pathogenic and nonpatho- genic species of mycobacteria can provide answers to the unresolved question of how pathogens subvert or inhibit an effective response. We infected human monocyte derived macrophages (hMDMs) with different species of mycobacteria, in increasing order of pathogenicity, i.e. M. smegmatis, M. bovis BCG, and M. tuberculosis R179 that had been cultured in the absence of detergents. RNA was isolated post-infection and transcriptomic analysis using amplicons (Ampliseq) revealed 274 differentially expressed genes (DEGs) across three species, out of which we selected 19 DEGs for further validation. We used qRT-PCR to confirm the differential expression of 19 DEGs. We studied biological network through Ingenuity Pathway Analysis® (IPA) which revealed up-regulated pathways of the interferon and interleukin family related to the killing of M. smegmatis. Apart from interferon and interleukin family, we found one up-regulated (EIF2AK2) and two down-regulated (MT1A and TRIB3) genes as unique potential targets found by Ampliseq and qRT-PCR which may be involved in the intracellular mycobacterial killing. The roles of these genes have not previously been described in tuberculosis. Multiplex ELISA of culture supernatants showed increased host immune response toward M. smegmatis as compared to M. bovis BCG and M.tb R179. These results enhance our understanding of host immune response against M.tb infection.Publisher's versio

2020-12-11 Global transcriptomic investigation of the human macrophage response towards pathogenic/non-pathogenic mycobacteria

Thesis (PhD)--Stellenbosch University, 2019.Background:Tuberculosis (TB) is a major cause of infection-related mortalityworldwide. In 2017 an estimated 1.3 million people who were HIV-negative died of TB. An estimated 5-10% of infected individual develop active TB during their lifetime, while the remaining90% (of infected population) successfully control the bacteria. Also, some of the close household contacts of TB patients remain uninfected and healthy. Studying host immune response towards Mycobacterium tuberculosis(M. tb) can unfold the reason behind this enigma. Methods:We conducted a detailed investigation of in vitrohost response from human monocyte derived macrophages(hMDMs)towards different strains of mycobacteria(grown in detergent-freemedia), i.e. pathogenic (M. tbR179) andnon-pathogenic (M. smegmatisand M. bovisBCG). The host response was measured post-infection (at mRNA and protein levels) using AmpliSeq, quantitative real time polymerase chain reaction (qRT-PCR), multiplex ELISA (Luminex), intracellular mycobacterial survivaland cytotoxicity assay. Biological network analysis (ingenuity pathway analysis IPA) was performed to understand the gene regulatory networkinvolved in the pathophysiology associated with the host-immune system.Based on false discovery rate (FDR) and biological functions, we selected an inter-related gene family of interferon induced protein with tetratricopeptides (IFIT1, IFIT2 andIFIT3) from the list of 19 potential differentially expressed genes(DEGs)for knock-up (vector-based over-expression)/down experiments. This gene family is known to form a protein complex during viral infection to act against the antigen. Studyencompassing their role against bacteria is not well established.Therefore, we performed knocking-up of IFITsvia vector-based transfection and knocking-down via small interferingRNA (siRNA) approach to investigate their effect upon mycobacteria inside the host macrophages. Results:AmpliSeqanalysis found 19 DEGs at 12 hours post-infection across all three strains. We observed lower number of mycobacterial CFUs and higher host response (at both RNA and protein level) in hMDMs infected with M. smegmatisas compared to other two strains. Biological network analysis revealed interferon-interleukin associated signalling pathways as most prominent among the 19 differentially expressed genes.We found a differed host response towardsall three strains, which mayattributeto their pathogenicity. Messenger RNA and protein level comparisons at different time points, depicted strong role of interferon and interleukin associated gene network. This network was able to successfully counter M. smegmatisbut succumb to M. bovisBCG andM. tbR179. Most importantly, across all three strains, intra-cellular bacterial growth and survival measured through colony forming units (CFUs)decreased significantly upon knocking up of IFITs(IFIT1, IFIT2 andIFIT3),while we recordedan increase in CFUs upon knocking down ofIFITsin the host macrophages. Using multiplex ELISA, we found higher expression of key pro-inflammatory cytokines (i.e. IDO1, IFN-γ, IL-6, and IL-23) during knock-up (vector-based over-expression)of IFITsresulting in reduction of mycobacteria. Conclusion:Differentially expressed IFITs showed a strong effect against mycobacteria, which can be used as a promising therapeutic targetadjunct to anti-TB therapy. This knowledge will broaden the scope of host drug targets for resistance free bacteriostatic immuno-therapy.Agtergrond:Tuberkulose (TB) is ‘n hoofoorsaak van infeksieverwante sterftes wêreldwyd. ‘n Benaderde 1.3 miljoen MIV-negatiewe mense is in 2017 dood aan TB. ‘n Benaderde 5-10% van geïnfekteerde individue ontwikkel aktiewe TB in hul leeftyd, terwyl die oorblywende 90%(van die geïnfekteerde bevolking) die bakterie suksesvol beheer. Sommige huisgenote van TB pasiënte bly ook ongeïnfekteerd en gesond. Die rede vir hierdie enigma kan ontbloot word deur die gasheer immuunreaksie te bestudeer. Metodes:Ons het deeglik ondersoek ingestel na die in vitrogasheerreaksie (van menslike monosiet-afgeleide makrofae) op verskillende stamme van mikobakterieë (opgegroei in ontsmettingsmiddelvrye media), d.i. patogenies (R179) en nie-patogenies (M..smegmatisen M. bovisBCG). Die gasheer reaksie is na infeksie gemeet (boodskapper RNS en proteïenvlakke) met AmpliSeq, reële tyd PKR, veelvuldige baan ELISA (Luminex), biologiese netwerk analise (Ingenuity Pathway Analysis), intrasellulêre mikobakteriële oorlewing en sitotoksisiteit eksperimente. Ons het ‘n onderling-verwante geenfamilie van interferon geïnduseerde proteïen met tetratrigopeptiede (IFIT1, IFIT2 enIFIT3)gekies uit die lys van 19 moontlike verskillend-uitgedrukte gene vir ons oor-uitdrukking, en onderdrukking eksperimente. Hierdie geenfamilie is bekend daarvoor om ‘n proteïenkompleks te vorm om gedurende virale infeksie teen die antigeen op te tree. Hul rol in bakteriële infeksie is nie goed bevestig nie. Daarom het ons oor-uitdrukking van IFITsdeur vektor-gebaseerde transfeksie en onderdrukking deur klein onderdrukkende RNS (siRNA) uitgevoer om hul effek op mikobakterieë in gasheer makrofae te ondersoek. Resultate:AmpliSeq analise het 19 verskillend-uitgedrukte gene teen 12 ure na infeksie oor al drie stamme gevind. Ons het ‘n laer getal kolonie-vormende eenhede en ‘n hoër gasheerreaksie (in beide RNS-en proteïenvlak) in menslike monosiet-afgeleide makrofae geïnfekteer met M. smegmatisopgemerk, as in die ander twee stamme. Biologiese netwerk analise het gewys dat interferon-interleuken verwante seinweë ‘n belangrike rol speel tussen die 19 verskillend-uitgedrukte gene.Ons het verskillende gasheerreaksies teenoor die 3 stamme gevind, wat moontlik aan elk se patogeniese aard verwant is. Boodskapper RNS en proteïenvlak vergelykings by verskillende tydpunte, het die belangrike rol van ‘ninterferon en interleuken verwante geennetwerk gewys. Hierdie netwerk kon M. smegmatissuksesvol beheer, maar nie M. bovisBCG en R179 nie.Interessant genoeg, het intra-sellulêre bakteriële groei en oorlewing, soos gemeet in kolonie-vormende eenhede, beduidend verminder wanneer IFITs oor-uitgedruk is, terwyl dit vermeerder het wanneer IFIT1, IFIT2 enIFIT3onderdruk is in gasheer makrofae. Intrasellulêre groei en oorlewing van mikobakterieë is bevestig deur boodskapper RNS uitdrukking met kwantifiserende PKR en proteïen uitdrukking met “Western blot”, te ondersoek. ‘n Paar sleutel-molekules is opgemerk in die sel sitokien uitdrukking wat ons met veelvuldige-baan ELISA ondersoek het. Dit het IDO1, IFN-γ, IL-6, en IL-23 ingesluit, wat bekend is as pro-inflammatoriese sitiokiene. Afsluiting:Hierdie uitslae sal ons kennis verbreed oor gasheer middel-teikens vir weerstandvrye bakteriostatiese immuunterapie aanvullend tot die huidige chemoterapie.Doctora

Study of dielectric relaxation and dipole moment of some hydrogen bonded solvent binary mixtures in 1,4-dioxane

943-952The dielectric relaxation and dipole moment of different concentration binary mixtures of mono methyl, mono ethyl and mono butyl ethers of ethylene glycol with four polar solvents i.e. ethyl alcohol, glycerol, dimethyl sulphoxide and dimethyl formamide have been measured in dilute solutions of 1,4-dioxane at 35°C. The measured values of permittivity (') and dielectric loss ('') at 10.1 GHz, static dielectric constant (o) at 1 MHz and high frequency limiting dielectric constant (∞) have been used to determine the values of molecular relaxation time ( o), and dipole moment ( ) of the different composition binary mixtures. The analysis of the results based on hetero-association in these binary systems, indicates the existence of different characteristic heterogeneous species in dilute solutions. The comparative values of o and of the various binary mixtures of different composition also suggest that the nature of heterogeneous interaction varies with the type of polar solvent and also constituents composition in the binary mixture

Comparison of human monocyte derived macrophages and THP1-like macrophages as in vitro models for M. tuberculosis infection

CITATION: Madhvi, A., et al. 2019. Comparison of human monocyte derived macrophages and THP1-like macrophages as in vitro models for M. tuberculosis infection. Comparative Immunology, Microbiology and Infectious Diseases, 67:101355, doi:10.1016/j.cimid.2019.101355.The original publication is available at https://www.sciencedirect.comMacrophages are the preferential cell types to study various aspects of mycobacterial infection. Commonly used infection models for in-vitro studies are primary macrophages such as human monocyte derived macrophages (hMDMs) and macrophage like cell lines (THP-1). It is not clear if commercially available THP-1 cells can be used as hMDMs alternative for in-vitro M.tb infection experiments. We conducted a detailed investigation of the hMDM and THP-1 response to mycobacterial infection on a comparative basis and assess the most crucial aspects of infection which are most commonly studied. We assessed mycobacterial uptake and intracellular growth over time of a pathogenic drug-resistant and drug-susceptible M.tb strains (R179 and H37Rv) through colony forming units (CFUs). Both strains depicted similar uptake and intracellular growth in hMDMs and THP-1 macrophages over time (R179, p=0.954) (H37Rv, p=0.922). Cytotoxicity assays revealed a consistent viability up to day 16 post-infection across the strains in both THP-1 and hMDMs (R179, p=0.271) (H37Rv, p=0.068). Interestingly, both cell lines showed similar mycobacterial uptake and cellular viability in both susceptible as well as resistant M.tb strains. Cytokine/chemokine mRNA analysis through qPCR found no difference between cell types. Further, cytokine secretion measured through Luminex revealed no difference across the strains. Also, cytokine secretion analysis showed no difference in both cell lines across strains. In conclusion, our study shows that THP-1 and hMDMs bacterial uptake, viability and host response to drug-susceptible and drug-resistant mycobacterial infections are similar. Therefore, present study demonstrate that THP-1 cells are suitable substitutes for hMDMs for in-vitro M.tb infection experiments.https://www.sciencedirect.com/science/article/pii/S0147957119301523Publisher's versio

Endoplasmic Reticulum Stress Induced Synthesis of a Novel Viral Factor Mediates Efficient Replication of Genotype-1 Hepatitis E Virus.

Hepatitis E virus (HEV) causes acute hepatitis in many parts of the world including Asia, Africa and Latin America. Though self-limiting in normal individuals, it results in ~30% mortality in infected pregnant women. It has also been reported to cause acute and chronic hepatitis in organ transplant patients. Of the seven viral genotypes, genotype-1 virus infects humans and is a major public health concern in South Asian countries. Sporadic cases of genotype-3 and 4 infection in human and animals such as pigs, deer, mongeese have been reported primarily from industrialized countries. Genotype-5, 6 and 7 viruses are known to infect animals such as wild boar and camel, respectively. Genotype-3 and 4 viruses have been successfully propagated in the laboratory in mammalian cell culture. However, genotype-1 virus replicates poorly in mammalian cell culture and no other efficient model exists to study its life cycle. Here, we report that endoplasmic reticulum (ER) stress promotes genotype-1 HEV replication by inducing cap-independent, internal initiation mediated translation of a novel viral protein (named ORF4). Importantly, ORF4 expression and stimulatory effect of ER stress inducers on viral replication is specific to genotype-1. ORF4 protein sequence is mostly conserved among genotype-1 HEV isolates and ORF4 specific antibodies were detected in genotype-1 HEV patient serum. ORF4 interacted with multiple viral and host proteins and assembled a protein complex consisting of viral helicase, RNA dependent RNA polymerase (RdRp), X, host eEF1α1 (eukaryotic elongation factor 1 isoform-1) and tubulinβ. In association with eEF1α1, ORF4 stimulated viral RdRp activity. Furthermore, human hepatoma cells that stably express ORF4 or engineered proteasome resistant ORF4 mutant genome permitted enhanced viral replication. These findings reveal a positive role of ER stress in promoting genotype-1 HEV replication and pave the way towards development of an efficient model of the virus

Deletion mapping of interaction domains in ORF4 for binding with g-1 ORF3, X and Helicase.

<p>Deletion mapping of interaction domains in ORF4 for binding with g-1 ORF3, X and Helicase.</p

ORF4 antibody is detected in HEV patients and its over expression enhances viral replication.

<p><b>(A)</b> Top: Purified GST-ORF4 stained with coomassie blue (left most) and western of equal aliquots of the same using healthy (CS1, CS2) and acute HEV infected (KU168733-KU168737) patient sera. Bottom: Top blots reprobed with anti-ORF4 antibody. <b>(B)</b> Western of whole cell extract from indicated cells using ORF4 and GAPDH antibodies. <b>(C)</b> QRT-PCR of sense and anti-sense RNA in pCDNA5-Huh7 and ORF4-Huh7 cells transfected with <i>in vitro</i> synthesized wild type (WT) or mutant HEV genome and treated as indicated.<b>(D)</b> Quantitation of viral ORF1 (helicase) and ORF2 expression in pCDNA5-Huh7 and ORF4-Huh7 cells, transfected with wildtype (WT HEV) or ORF4 expression deficient mutant (DM HEV) HEV genomic RNA and treated with the indicated compounds. Ten random fields of approximately 30 cells in each field were counted for helicase, ORF2, DAPI (nuclear stain) fluorescence and percentage ±SEM calculated.</p

Yeast Two Hybrid screening of host factors that directly interact with g-1 RdRp and ORF4.

<p>Yeast Two Hybrid screening of host factors that directly interact with g-1 RdRp and ORF4.</p

An IRESl (Internal ribosome entry site-like) element located upstream of ORF4 coding sequence drives its translation independent of ORF1.

<p><b>(A)</b> Predicted secondary structure of IRESl using “mfold”. Cyan letters indicate core IRESl sequence. A, B, C: stem loops, A*: bulge. Substitutions that impair IRESl are in bold. <b>(B)</b> Organization of Dual luciferase reporter plasmid. <b>(C)</b> Dual luciferase reporter assay. Values are mean±SEM.</p

ORF4 is a target of host ubiquitin-proteasome machinery.

<p><b>(A)</b> Western blot using anti-Flag (top) and anti-GAPDH (bottom). <b>(B)</b> Western blot using anti-ubiquitin (top) and anti-Flag (bottom). <b>(C)</b> Anti-Flag western blot of Huh7 cells expressing WT and K51N mut ORF4, treated with cycloheximide (panel 1, 3). Same blots were reprobed with anti-GAPDH (panel 2, 4). <b>(D)</b> Immunofluorescence of ORF4 in Huh7 cells transfected with <i>in vitro</i> synthesized WT HEV or K51N HEV. Scale: 20μm. Shown are merged images of nuclei (blue) and ORF4 (green). “→”: positive staining, “►”: unstained. <b>(E)</b> QRT-PCR of HEV sense and anti-sense RNA from Huh7 cells transfected and treated as indicated. Veh: Vehicle, TUN:tunicamycin.</p