Immunopathology of and potential therapeutics for secondary hemophagocytic lymphohistiocytosis/macrophage activation syndrome: a translational perspective.

Secondary hemophagocytic lymphohistiocytosis/macrophage activation syndrome (sHLH/MAS) is a life-threatening immune disorder triggered by rheumatic disease, infections, malignancies, or medications. Characterized by the presence of hemophagocytic macrophages and a fulminant cytokine storm, sHLH/MAS leads to hyperferritinemia and multiorgan failure and rapidly progresses to death. The high mortality rate and the lack of specific treatments necessitate the development of a new drug. However, the complex and largely unknown immunopathologic mechanisms of sHLH/MAS, which involve dysfunction of various immune cells, diverse etiologies, and different clinical contexts make this effort challenging. This review introduces the terminology, diagnosis, and clinical features of sHLH/MAS. From a translational perspective, this review focuses on the immunopathological mechanisms linked to various etiologies, emphasizing potential drug targets, including key molecules and signaling pathways. We also discuss immunomodulatory biologics, existing drugs under clinical evaluation, and novel therapies in clinical trials. This systematic review aims to provide insights and highlight opportunities for the development of novel sHLH/MAS therapeutics.

Tryptophanyl-tRNA Synthetase 1 Signals Activate TREM-1 via TLR2 and TLR4.

Tryptophanyl-tRNA synthetase 1 (WARS1) is an endogenous ligand of mammalian Toll-like receptors (TLR) 2 and TLR4. Microarray data, using mRNA from WARS1-treated human peripheral blood mononuclear cells (PBMCs), had indicated WARS1 to mainly activate innate inflammatory responses. However, exact molecular mechanism remains to be understood. The triggering receptor expressed on myeloid cells (TREM)-1 is an amplifier of pro-inflammatory processes. We found WARS1 to significantly activate TREM-1 at both mRNA and protein levels, along with its cell surface expression and secretion in macrophages. WARS1 stimulated TREM-1 production via TLR2 and TLR4, mediated by both MyD88 and TRIF, since targeted deletion of TLR4, TLR2, MyD88, and TRIF mostly abrogated TREM-1 activation. Furthermore, WARS1 promoted TREM-1 downstream phosphorylation of DAP12, Syk, and AKT. Knockdown of TREM-1 and inhibition of Syk kinase significantly suppressed the activation of inflammatory signaling loop from MyD88 and TRIF, leading to p38 MAPK, ERK, and NF-κB inactivation. Finally, MyD88, TRIF, and TREM-1 signaling pathways were shown to be cooperatively involved in WARS1-triggered massive production of IL-6, TNF-α, IFN-ß, MIP-1α, MCP-1, and CXCL2, where activation of Syk kinase was crucial. Taken together, our data provided a new insight into WARS1's strategy to amplify innate inflammatory responses via TREM-1.

Cortactin Interacts with Hepatitis C Virus Core and NS5A Proteins: Implications for Virion Assembly.

Hepatitis C virus (HCV) exploits cellular proteins to facilitate viral propagation. To identify the cellular factors involved in the HCV life cycle, we previously performed protein microarray assays using either HCV nonstructural 5A (NS5A) protein or core protein as a probe. Interestingly, cellular cortactin strongly interacted with both NS5A and core. Cortactin is an actin-binding protein critically involved in tumor progression by regulating the migration and invasion of cancerous cells. Protein interaction between cortactin and NS5A or core was confirmed by coimmunoprecipitation and immunofluorescence assays. We showed that cortactin interacted with NS5A and core via the N-terminal acidic domain of cortactin. Cortactin expression levels were not altered by HCV infection. Small interfering RNA (siRNA)-mediated knockdown of cortactin dramatically decreased HCV protein expression and infectivity levels, whereas overexpression of cortactin increased viral propagation. Ectopic expression of the siRNA-resistant cortactin recovered the viral infectivity, suggesting that cortactin was specifically required for HCV propagation. We further showed that cortactin was involved in the assembly step without affecting viral entry, HCV internal ribosome entry site (IRES)-mediated translation, and the replication steps of the HCV life cycle. Of note, silencing of cortactin markedly reduced both NS5A and core protein levels on the lipid droplets (LDs), and this effect was reversed by the overexpression of cortactin. Importantly, NS5A and core promoted cell migration by activating the phosphorylation of cortactin at tyrosine residues 421 and 466. Taken together, these data suggest that cortactin is not only involved in HCV assembly but also plays an important role in the cell migration.IMPORTANCE Cortactin is a cytoskeletal protein that regulates cell migration in response to a number of extracellular stimuli. The functional involvement of cortactin in the virus life cycle is not yet fully understood. The most significant finding is that cortactin strongly interacted with both hepatitis C virus (HCV) core and NS5A. Cortactin is involved in HCV assembly by tethering core and NS5A on the lipid droplets (LDs) with no effect on LD biogenesis. It was noteworthy that HCV NS5A and core activated cortactin by phosphorylation at tyrosines 421 and 466 to regulate cell migration. Collectively, our study shows that cortactin is a novel host factor involved in viral production and HCV-associated pathogenesis.

Hepatitis C Virus Downregulates Ubiquitin-Conjugating Enzyme E2S Expression To Prevent Proteasomal Degradation of NS5A, Leading to Host Cells More Sensitive to DNA Damage.

Hepatitis C virus (HCV) infection may cause chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV exploits cellular machineries to establish persistent infection. We demonstrate here that ubiquitin-conjugating enzyme E2S (UBE2S), a member of the ubiquitin-conjugating enzyme family (E2s), was downregulated by endoplasmic reticulum stress caused by HCV in Huh7 cells. UBE2S interacted with domain I of HCV NS5A and degraded NS5A protein through the Lys11-linked proteasome-dependent pathway. Overexpression of UBE2S suppressed viral propagation, while depletion of UBE2S expression increased viral infectivity. Enzymatically inactive UBE2S C95A mutant exerted no antiviral activity, suggesting that ubiquitin-conjugating enzymatic activity was required for the suppressive role of UBE2S. Chromatin ubiquitination plays a crucial role in the DNA damage response. We showed that the levels of UBE2S and Lys11 chains bound to the chromatin were markedly decreased in the context of HCV replication, rendering HCV-infected cells more sensitive to DNA damage. These data suggest that HCV counteracts antiviral activity of UBE2S to optimize viral propagation and may contribute to HCV-induced liver pathogenesis.IMPORTANCE Protein homeostasis is essential to normal cell function. HCV infection disturbs the protein homeostasis in the host cells. Therefore, host cells exert an anti-HCV activity in order to maintain normal cellular metabolism. We showed that UBE2S interacted with HCV NS5A and degraded NS5A protein through the Lys11-linked proteasome-dependent pathway. However, HCV has evolved to overcome host antiviral activity. We demonstrated that the UBE2S expression level was suppressed in HCV-infected cells. Since UBE2S is an ubiquitin-conjugating enzyme and this enzyme activity is involved in DNA damage repair, HCV-infected cells are more sensitive to DNA damage, and thus UBE2S may contribute to viral oncogenesis.

Hepatitis C Virus Modulates Solute carrier family 3 member 2 for Viral Propagation.

Hepatitis C virus (HCV) exploits an extensive network of host proteins to maintain chronic infection. Using RNA-Seq technology, we identified 30 host genes that were differentially expressed in cell culture grown HCV (HCVcc)-infected cells. Of these candidate genes, we selected solute carrier family 3 member 2 (SLC3A2) for further investigation. SLC3A2, also known as CD98hc, is a member of the solute carrier family and encodes a subunit of heterodimeric amino acid transporter. SLC3A2 and LAT1 constitute a heterodimeric transmembrane protein complex that catalyzes amino acid transport. In this study, we showed that HCV upregulated both mRNA and protein expression levels of SLC3A2 and this upregulation occurred through NS3/4A-mediated oxidative stress. HCV also elevated SLC3A2/LAT1 complex level and thus mammalian target of rapamycin complex 1 (mTORC1) signaling was activated. We further showed that L-leucine transport level was significantly increased in Jc1-infected cells as compared with mock-infected cells. Using RNA interference technology, we demonstrated that SLC3A2 was specifically required for the entry step but not for other stages of the HCV life cycle. These data suggest that SLC3A2 plays an important role in regulating HCV entry. Collectively, HCV exploits SLC3A2 for viral propagation and upregulation of SLC3A2 may contribute to HCV-mediated pathogenesis.

Nonstructural Protein 5A Impairs DNA Damage Repair: Implications for Hepatitis C Virus-Mediated Hepatocarcinogenesis.

RAD51-associated protein 1 (RAD51AP1) is a member of the multiprotein complexes postulated to carry out RAD51-mediated homologous recombination and DNA repair in mammalian cells. In the present study, we showed that hepatitis C virus (HCV) NS5A directly bound RAD51AP1 and increased the protein level of RAD51AP1 through modulation of the ubiquitin-proteasome pathway. We also demonstrated that RAD51AP1 protein levels were increased in the liver tissues of HCV-infected patients and NS5A-transgenic mice. Importantly, NS5A impaired DNA repair by disrupting the RAD51/RAD51AP1/UAF1 complex and rendered HCV-infected cells more sensitive to DNA damage. Silencing of RAD51AP1 expression resulted in a decrease of viral propagation. We further demonstrated that RAD51AP1 was involved in the assembly step of the HCV life cycle by protecting viral RNA. These data suggest that HCV exploits RAD51AP1 to promote viral propagation and thus that host DNA repair is compromised in HCV-infected cells. Overall, our findings provide mechanistic insight into the pathogenesis of HCV infection.IMPORTANCE Chronic infection with HCV is the leading cause of hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying HCV-induced HCC are not fully understood. Here we demonstrate that the HCV NS5A protein physically interacts with RAD51AP1 and increases the RAD51AP1 protein level through modulation of the ubiquitin-proteasome pathway. HCV coopts host RAD51AP1 to protect viral RNA at an assembly step of the HCV life cycle. Note that the RAD51 protein accumulates in the cytoplasm of HCV-infected cells, and thus the RAD51/RAD51AP1/UAF1-mediated DNA damage repair system in the nucleus is compromised in HCV-infected cells. Our data may provide new insight into the molecular mechanisms of HCV-induced pathogenesis.

Rad51 Interacts with Non-structural 3 Protein of Hepatitis C Virus and Regulates Viral Production.

Hepatitis C virus (HCV) is a leading cause of chronic liver disease affecting over 170 million people worldwide. Chronic infection with HCV progresses to liver fibrosis, cirrhosis, and hepatocellular carcinoma. HCV exploits host cellular factors for viral propagation. To investigate the cellular factors required for HCV propagation, we screened a siRNA library targeting human cell cycle genes using cell culture grown HCV-infected cells. In the present study, we selected and characterized a gene encoding Rad51. Rad51, a member of a conserved recombinase family, is an essential factor for homologous recombination and repair of double-strand DNA breaks. We demonstrated that siRNA-mediated knockdown of Rad51 significantly inhibited HCV propagation without affecting HCV RNA replication. Silencing of Rad51 impaired secretion of infectious HCV particles and thus intracellular viruses were accumulated. We showed that HCV NS3 specifically interacted with Rad51 and accumulated Rad51 in the cytosol. Furthermore, Rad51 was coprecipitated with NS3 and HCV RNA. By employing membrane flotation and protease protection assays, we also demonstrated that Rad51 was co-fractionated with HCV NS3 on the lipid raft. These data indicate that Rad51 may be a component of the HCV RNA replication complex. Collectively, these data suggest that HCV may exploit cellular Rad51 to promote viral propagation and thus Rad51 may be a potential therapeutic target for HCV.

MARCO variants are associated with phagocytosis, pulmonary tuberculosis susceptibility and Beijing lineage.

Macrophage receptor with collagenous structure (MARCO) has an important role in the phagocytosis of Mycobacterium tuberculosis (M. tuberculosis). We hypothesized that MARCO polymorphisms are associated with phagocytosis, tuberculosis (TB) disease susceptibility and presentation, and infecting lineage. We used a human cellular model to examine how MARCO genotype mediates the immune response; a case-control study to investigate tuberculosis host genetic susceptibility; and a host-pathogen genetic analysis to study host-pathogen interactions. Two MARCO heterozygous (AG) genotypes (single-nucleotide polymorphisms rs2278589 and rs6751745) were associated with impaired phagocytosis of M. tuberculosis trehalose 6,6'-dimycolate-cord factor and ß-glucan-coated beads in macrophages. The heterozygous genotypes of rs2278589 and rs6751745 were also associated with increased risk of pulmonary TB (PTB; rs2278589, P=0.001, odds ratio (OR)=1.6; rs6751745, P=0.009, OR=1.4), and with severe chest X-ray abnormalities (P=0.007, OR=1.6). These two genotypes were also associated with the Beijing lineage (rs2278589, P=0.001, OR=1.7; rs6751745, P=0.01, OR=1.5). Together, these results suggest that MARCO polymorphisms may regulate phagocytosis of M. tuberculosis and susceptibility and severity of PTB. They also suggest MARCO genotype and Beijing strains may interact to increase the risk of PTB.

Development of ARI case management at primary and secondary level in southern Vietnam.

In southern Vietnam it is not uncommon that children under 5 years of age die from pneumonia. Reduction of severity and mortality has to rely on proper case management by mothers and health workers on both grass root level and referral level. The responsibility of training of clinical skills of ARI case management in the southern provinces of Vietnam has been delegated to Pediatric Hospital N1 (PHN1) Ho Chi Minh City (HCMC) by Ministry of Health. A pilot project was carried out by the Danish-Vietnamese Study Group. The immediate objects were: to provide basic epidemiological information about ARI in southern Vietnam, to develop training modules and case management intervention modules at primary and secondary level in order to enable mothers, village workers, health post staff and district hospital emergency department staff to treat moderate and severe pneumonia and acute bronchitis in accordance with the WHO management guide for ARI and to evaluate the effect of those modules after implementation in a limited number of communes. The modules were developed at PHN1. Ten commune health stations were carefully selected. The purpose of the project and the conditions for taking part had been explained to the health workers. The doctors and other commune health workers from the 10 commune health stations and doctors from the connected district hospitals attended the training courses at PHN1, HCMC and also at the belonging provincial hospitals. Essential equipment was provided and a pharmacy with essential drugs established. The registered health statistics was collected yearly during on site visits. The local doctors and commune health workers gave seminars for mothers in the villages of the 10 project communes. The mothers' knowledge, attitude and practice (KAP) was tested in interviews before and two months after the seminars had taken place. The spread of KAP was measured by random interviews of mothers six month later. In the interviews information on social conditions was obtained. The mothers' KAP had risen by 25% two months after attending the seminars. A further increase of KAP by 5-10% within the untrained group appeared in a survey 4-6 months later. It was not possible to obtain reliable statistics on morbidity or mortality of ARI in the project area.

Integration of primary health care concepts in a children's hospital with limited resources.

After nearly 30 years of war, health services in Vietnam were devastated. Pediatric Hospital Number 1 (PH1) in Ho Chi Minh City was severely overloaded, mortality rates for readily treatable diseases were high, and staff competence and motivation were low. In 1988, PH1 introduced primary health care (PHC) concepts into the policy of the hospital. The approach included identification of priority diseases that are most easily treatable (diarrhoea, acute respiratory infections, Dengue haemorrhagic fever, malnutrition, and common paediatric emergencies including malaria); establishment of training programmes through paediatric priority training units for medical staff of PH1 and health centres (district and commune services), and health education for the patients' carers; promotion of outpatient treatment to avoid unnecessary admissions; use of appropriate technology such as essential drugs and application of WHO guidelines; support for health centres; transfer of responsibility for decision-making from one central authority to each department; and community participation, by which we sought small contributions from families who could afford to pay. Since the new approach was implemented, the numbers of admissions have fallen substantially. Mortality rates have decreased greatly (diarrhoea by 80%, Dengue fever by 64%, and acute respiratory infections by 41%). Support from foreign non-governmental organisations has enabled training and research to enhance staff skill and knowledge and supply of necessary equipment.(ABSTRACT TRUNCATED AT 250 WORDS)