Naglaa H Shoukry

• B.Pharm, PhD
• Professor (Associate) at Université de Montréal

Liver fibrosis is a global health problem. IL-17A has proven profibrogenic properties in liver disease making it an interesting therapeutic target. IL-17A is regulated by RORγt and produced by Th17 CD4+ and γδ-T cells. We hypothesized that blocking IL-17A production will limit fibrosis progression by reducing recruitment of inflammatory cells. Here...

Identification and isolation of antigen-specific T cells for downstream transcriptomic analysis is key for various immunological studies. Traditional methods using major histocompatibility complex (MHC) multimers are limited by the number of predefined immunodominant epitopes and MHC matching of the study subjects. Activation-induced markers (AIM)...

Metabolic dysfunction-associated steatotic liver disease (MASLD) comprises a spectrum of liver diseases that span simple steatosis, metabolic dysfunction-associated steatohepatitis (MASH) and fibrosis and may progress to cirrhosis and cancer. The pathogenesis of MASLD is multifactorial and is driven by environmental, genetic, metabolic and immune f...

Introduction Follicular helper T cells are essential for helping in the maturation of B cells and the production of neutralizing antibodies (NAbs) during primary viral infections. However, their role during recall responses is unclear. Here, we used hepatitis C virus (HCV) reinfection in humans as a model to study the recall collaborative interacti...

The human immunodeficiency virus (HIV) persists in HIV viral reservoirs despite antiretroviral therapy (ART). Hepatitis C virus (HCV) coinfection is associated with increased HIV reservoir size and residual HIV transcription during ART. Herein, we investigated the impact of direct acting antivirals (DAA)-mediated HCV cure on the size/transcriptiona...

The liver is situated at the interface of the gut and circulation where it acts as a filter for blood-borne and gut-derived microbes and biological molecules, promoting tolerance of non-invasive antigens while driving immune responses against pathogenic ones. Liver resident immune cells such as Kupffer cells (KCs), a subset of macrophages, maintain...

Controlled human infection model trials for hepatitis C virus represent an important opportunity to identify correlates of protective immunity against a well-characterized inoculum of hepatitis C virus and how such responses are modified by vaccination. In this article, we discuss the approach to immunological monitoring during such trials, includi...

Successive episodes of hepatitis C virus (HCV) infection represent a unique natural rechallenge experiment to define correlates of long-term protective immunity and inform vaccine development. We applied a systems immunology approach to characterize longitudinal changes in the peripheral blood transcriptomic signatures in eight subjects who spontan...

SUPPLEMENTARY FIGURE S1 Ki67 levels are highest at 48 h post-CCl4. Representative Ki67 IHC images at 0, 24, 48, 72, 96 and 168 hours post -CCl4, scale bar = 200 mm.

SUPPLEMENTARY FIGURE S2 Circulating myeloid cells are recruited to the liver in response to CCl4 acute injury during the necroinflammatory and tissue repair phases. (A) Gating strategy for intrahepatic phagocyte. (B) Representative pseudocolor plots of the kinetics of major phagocyte populations induced in response to CCl4 injury. N=4-5 mice per gr...

SUPPLEMENTARY FIGURE S5 IBA1+ CLEC4F- macrophages and IBA1+ CLEC4F+ KCs are present in murine fibrotic liver. (A) Multiplex IF showing IBA1 (red) and CLEC4F (green) in liver sections from 12 weeks CCl4- treated mouse. Colocalization of red and green shows as yellow, scale bar = 200 mm, * designates CVs. (B) Higher magnifications of insets I, II, an...

SUPPLEMENTARY VIDEO 1 Activated hepatic stellate cells, IBA1+CLEC4F- macrophages and CLEC4F+ KCs establish direct contact around injured central veins at 48 h post- CCl4. Three-dimensional (3D) reconstruction of the interaction between an activated HSC in red (based on aSMA signal), an IBA1+CLEC4Fmacrophage in yellow (based on IBA1 signal), and a C...

SUPPLEMENTARY FIGURE S6 Globular shaped IBA1+ cells infiltrate the necrotic tissue at 24 h and replace hepatocytes in the area surrounding CVs by 48 h post-CCl4. Aligned serial sections of hepatic tissue: section 1 (H&E) and section 2 (IBA1 IHC). Red arrowheads pointing at infiltrating amoeboid IBA1+ macrophages at 24 h post-CCl4, scale bar= 50 mm....

SUPPLEMENTARY FIGURE S3 Recovered CD11b+ MHCII+ CD64+ F4/80+ cells express CX3CR1, and therefore are not KCs. (A) Gating strategy to define KCs from total intrahepatic leukocytes. (B) Contour plots showing CX3CR1 expression by total myeloid cells (left column), by CD11b+ MHCII+ CD64+ F4/80+ cells (center column), and the merged overlay (right colum...

SUPPLEMENTARY FIGURE S4 IBA1+CLEC4F- macrophages and CLEC4F+ KCs exhibit different spatial profiles in response to CCl4-induced acute liver injury. (A) Representative IBA1 (red) and CLEC4F (yellow) IF images of whole tissue at 48 hours post CCl4 at low magnification, scale bar = 1 mm. (B) High magnification of inset from A) showing DAPI/IBA1 (left)...

Background & Aims Non-alcoholic fatty liver disease (NAFLD) is a major health problem with complex pathogenesis. Although sex difference in NAFLD pathogenesis has been reported, the mechanisms underlying such difference remain understudied. IL-22 is a pleiotropic cytokine with both protective and/or pathogenic effects during liver injury. IL-22 was...

Non-alcoholic fatty liver disease (NAFLD) affects approximately 8 million Canadians. NAFLD refers to a disease spectrum ranging from bland steatosis to non-alcoholic steatohepatitis (NASH). Nearly 25% of patients with NAFLD develop NASH, which can progress to liver cirrhosis and related end-stage complications. Type 2 diabetes and obesity represent...

The hepatitis B and C viruses persist by evasion of T cell immunity. Persistence depends upon premature failure of CD4+ T cell help and loss of CD8+ T cell control because of epitope mutational escape and/or functional exhaustion. Powerful new immunological and transcriptomic tools provide insight into the mechanisms of T cell silencing by HBV and...

Nearly 2.3 million individuals worldwide are coinfected with human immunodeficiency virus (HIV) and hepatitis C virus (HCV). Odds of HCV infection are six times higher in people living with HIV (PLWH) compared to their HIV-negative counterparts, with the highest prevalence among people who inject drugs (PWID) and men who have sex with men (MSM). HI...

Hepatitis C virus (HCV) infection resolves spontaneously in ∼25% of acutely infected humans where viral clearance is mediated primarily by virus-specific CD8+ T cells. Previous cross-sectional analysis of the CD8+ TCR repertoire targeting two immunodominant HCV epitopes reported widespread use of public TCRs shared by different subjects, irrespecti...

Response to immune checkpoint blockade cancer immunotherapy is variable, but the mechanisms that underlie this variability remain unclear. In a recent issue of Nature Medicine, Yu et al. demonstrate that liver metastases limit immunotherapy efficacy by promoting macrophage-mediated elimination of tumor-specific CD8⁺ T cells. Liver-directed radiothe...

Early appearance of neutralizing antibodies during acute hepatitis C virus (HCV) infection is associated with spontaneous viral clearance. However, the longitudinal changes in antigen-specific memory B cell (MBCs) associated with divergent HCV infection outcomes remain undefined. We characterized longitudinal changes in E2 glycoprotein-specific MBC...

Importance Achievement of the World Health Organization (WHO) target of eliminating hepatitis C virus (HCV) by 2030 will require an increase in key services, including harm reduction, HCV screening, and HCV treatment initiatives in member countries. These data are not available for Canada but are important for informing a national HCV elimination s...

Hepatitis C virus (HCV) affects approximately 250,000 Canadians. Although safe and effective (>95% cure rates) antiviral therapies have become available within the past 5 years, chronic HCV infection still remains a major driver of end-stage liver disease and liver transplantation. Both the Canadian Institute for Health Research and the Public Heal...

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density and the location of immune cells in the TME have important diagnostic and prognostic values. Multiomic profiling of the TME has exponentially increased our understanding of the numerous cellular a...

Transfer of hepatitis C virus (HCV) infection from a donor to a new recipient is associated with a bottleneck of genetic diversity in the transmitted viral variants. Existing data suggests that one, or very few, variants emerge from this bottleneck to establish the infection (transmitted founder [T/F] variants). In HCV, very few T/F variants have b...

Background: While opioid agonist therapy (OAT) reduces the risk of HCV acquisition among people who inject drugs (PWID), protective effects may be attenuated in females compared to males. This study assessed sex disparities in HCV incidence among PWID exposed to OAT and factors independently associated with decreased protective efficacy. Methods:...

The engagement of activating NK receptors (aNKR) stimulates NK cell activity, provided that interactions between inhibitory NK receptors (iNKR) with their HLA ligands do not override them. Abs bound to target cells can also activate NK cells by engaging the CD16 aNKR. NK cell education status is an important factor for Ab‐dependent NK cell activati...

Interactions between the host immune system and the viral variants determine persistence of hepatitis C virus (HCV) infection after the acute phase of infection. This study describes the genetic variability of within‐host HCV viral variants in acute infection and correlates it with host and virus‐related traits and infection outcome. Next generatio...

Inflammatory immune cells can modulate activation of hepatic stellate cells (HSCs) and progression of liver fibrosis. Type 3 inflammation characterized by production of interleukin-17A (IL-17) and IL-22 by innate and adaptive immune cells is implicated in many inflammatory conditions of the gut and can be counteracted by regulatory T cells (T regs...

Most individuals exposed to hepatitis C virus (HCV) become persistently infected while a minority spontaneously eliminate the virus. Although early immune events influence infection outcome, the cellular composition, molecular effectors, and timeframe of the host response active shortly after viral exposure remain incompletely understood. Employing...

Hepatitis C virus (HCV) affects more than 268,000 people in Canada. Both the Canadian Institutes of Health Research and the Public Health Agency of Canada recognize the significant impact of HCV-related liver diseases and supported the establishment of a national hepatitis C research network, the Canadian Network on Hepatitis C (CanHepC). Interfero...

The type 3 cytokines IL-17 and IL-22 play a crucial, well synchronized physiological role in wound healing and repairing tissue damage due to infections or injury at barrier surfaces. These cytokines act on epithelial cells to induce secretion of early immune mediators, recruitment of inflammatory cells to the site of injury, and to trigger tissue...

The introduction of small molecules targeting viral functions has caused a paradigm shift in hepatitis C virus (HCV) treatment. Administration of these direct-acting antivirals (DAAs) achieves a complete cure in almost all treated patients with short-duration therapy and minimal side effects. Although this is a major improvement over the previous p...

The development of vaccines that protect against persistent hepatitis C virus (HCV) infection remain a public health priority. The broad use of highly effective direct-acting antivirals (DAAs) is unlikely to achieve HCV elimination without vaccines that can limit viral transmission. Two vaccines targeting either the antibody or the T cell response...

Background Activation of hepatic stellate cells (HSCs) is a key event in the initiation of liver fibrosis. CD4 T cells can modulate positively or negatively this process. Briefly, Th1 cells despite their pro-inflammatory properties have anti-fibrogenic properties in contrast to Th2 cells. We and others have demonstrated that IL-17A produced by Th17...

Background Hepatitis C virus infection becomes chronic in most cases while a minority (25%) can spontaneously resolve the infection. Antibodies that are specific to the envelope glycoproteins E1 and E2 are generated late during acute infection and yet their role in spontaneous clearance remains debated. Some reports described cases of viral clearan...

Chronic viral infections remain a global health concern. The early events that facilitate viral persistence have been linked to the activity of the immunoregulatory cytokine IL-10. However, the mechanisms by which IL-10 facilitates the establishment of chronic infection are not fully understood. Herein, we demonstrated that the antigen sensitivity...

Inflammatory bowel disease (IBD) involves interaction between host genetic factors and environmental triggers. CCDC88B maps within one IBD risk locus on human chromosome 11q13. Here we show that CCDC88B protein increases in the colon during intestinal injury, concomitant with an influx of CCDC88B⁺lymphoid and myeloid cells. Loss of Ccdc88b protects...

Background: The objective of this study was to assess differences in hepatitis C virus (HCV) incidence by sex in people who inject drugs (PWID), using a large international multi-cohort set of pooled biological and behavioral data from prospective observational studies of incident HIV and HCV infections in high-risk cohorts (the InC3 Collaborative...

Nonalcoholic fatty liver disease (NAFLD) is now the most common progressive liver disease in developed countries and is the second leading indication for liver transplantation due to the extensive fibrosis it causes. NAFLD progression is thought to be tied to chronic low-level type 1 inflammation originating in the adipose tissue during obesity; ho...

Activation of hepatic stellate cells (HSCs) is a key event in the initiation of liver fibrosis. We and others have demonstrated that IL-17A produced by Th17 cells promotes activation of HSCs. Th17 cells also produce IL-22, an enigmatic cytokine with pro-inflammatory and hepatoprotective properties. In addition, regulatory T cells (Treg) negatively...

Background We determined temporal trends (1985–2011) in hepatitis C virus (HCV) incidence and associated behavioral exposures for people who inject drugs (PWID) from the United States (Boston, Baltimore, and San Francisco), Canada (Montreal), the Netherlands (Amsterdam), and Australia (Sydney and Melbourne). Methods Using population-based cohort d...

The dynamics of the memory CD8 T cell receptor (TCR) repertoire upon virus re-exposure and factors governing the selection of TCR clonotypes conferring protective immunity in real life settings are poorly understood. Here, we examined the dynamics and functionality of the virus-specific memory CD8 TCR repertoire before, during and after hepatitis C...

The gating strategy used for the sorting of different epitope-specific tetramer+ CD8+ T-cell populations. Pre-purified CD8 T cells were stained and sorted as viable CD3+ CD8+ tetramer positive cells, and when possible also according to the expression of CD127. Naive CD8 T cells were sorted as viable CD3+ CD8+ CD45RO-. (TIF)

Patients’ clinical characteristics and demographics. (DOCX)

TCR deep sequencing summary information. (DOCX)

Dominant clonotype (Freq > 1%) usage in B27/NS5B-2841-specific CD8 T cells for patient SR/SR-3 during HCV reinfection. (DOCX)

Purity of sorted T-cell populations used for TCR-repertoire analysis. Dot plots showing the post-sorting purity of the sorted (A) epitope-specific tetramer+ CD8+ T-cell population and (B) naïve CD8+ T cells (viable CD3+ CD8+ Tetramer-). (TIF)

HCV-specific tetramer+ CD8 T-cell clonotypes recruited during the reinfection episode were present at the peak of primary infection. The top ten dominant clonotypes (frequency ≥1%) isolated directly ex vivo from patient SR/SR-3 followed-up longitudinally during primary HCV infection and reinfection episode at pre-reinfection, peak expansion and pos...

CDR3 sequences characteristics do not differ between groups. Average nucleotides (NT) additions (A), CDR3 region length (B) and germline index (total repertoire (C) and dominant clonotypes only (D)) are shown for both patient groups at the three different time points. (TIF)

Dominant clonotype (Freq >1%) usage in A1/NS3-1436-specific CD8 T cells for patient SR/CI-3 during HCV reinfection. (DOCX)

CD8 T cell lines TCR deep sequencing. (DOCX)

Venn diagrams showing the number of unique clonotypes present for each time point and shared between time points. Clonotypes present only at the pre reinfection time point are located in the red circles, at peak reinfection in the green circle and post reinfection in the blue circle. Clonotypes shared between two or more time points are located at...

Higher polyfunctionality for resolvers SR/SR compared to chronic SR/CI T cell lines. T cell lines were stimulated with autologous BLCLs prepulsed with increasing concentrations of the cognate peptide (NS3-1073) for 6 h. Surface and intracellular staining was then performed as described in Materials and Methods to examine functionality by flowcytome...

Dominant clonotype (Freq ≥1%) usage in A2/NS3-1073 –specific CD8 T cells for patient SR/SR-1 during HCV reinfection. (DOCX)

Dominant clonotype (Freq ≥1%) usage in B27/NS5B-2841-specific CD8 T cells for patient SR/SR-2 during HCV reinfection. (DOCX)

Dominant clonotype (Freq > 1%) usage in A2/NS3-1073-specific CD8 T cells for patient SR/CI-2 during HCV reinfection. (DOCX)

Broadly neutralizing antibodies have been associated with spontaneous clearance of the hepatitis C infection as well as viral persistence by immune escape. Further study of neutralizing antibody epitopes is needed to unravel pathways of resistance to virus neutralization, and to identify conserved regions for vaccine design. All reported broadly ne...

Several direct-acting antivirals (DAAs) have been approved for the treatment of chronic hepatitis C virus (HCV) infections, opening the door to highly effective interferon-free treatment regimens. Resistance-associated substitutions (RASs) have been reported both in treatment-naïve patients and following treatment with protease (NS3), phosphoprotei...

The liver is an important metabolic organ with unique immunological properties. As a secondary lymphoid organ, the liver contains several populations of antigen-presenting cells including classical dendritic cells, but also hepatocytes, Kupffer cells, endothelial cells, and hepatic stellate cells that are able to uptake, process, and present antige...

The interferon (IFN)-λ family of type III cytokines includes the closely related interleukin (IL)-28A (IFN-λ2), IL-28B (IFN-λ3), and IL-29 (IFN-λ1). They signal through the Janus kinases (JAK)-signal transducers and activators of transcription pathway and promote an antiviral state by the induction of expression of several interferon-stimulated gen...

Acute hepatitis C virus (HCV) infection culminates in viral persistence in the majority of cases. Abs that recognize the envelope glycoproteins E1 and E2 are generated during the late stages of acute infection, yet their contribution to spontaneous viral clearance remains controversial. Investigation of the humoral responses during acute HCV infect...

Cross-continental phylogenetic analysis is important to understand subtle molecular differences of currently circulating hepatitis C virus (HCV) subtypes. Existence of such differences can be crucial in pursuing a universal hepatitis C vaccine. We characterized molecular epidemiology of early HCV infections identified across nine cohorts [North Ame...

Background: In Canada, interferon-free, direct-acting antiviral hepatitis C virus (HCV) regimens are costly. This presents challenges for universal drug coverage of the estimated 220 000 people with chronic HCV infection nationwide. The study objective was to appraise criteria for reimbursement of 4 HCV direct-acting antivirals in Canada. Methods...

Background: Approximately one quarter of persons exposed to hepatitis C virus (HCV) will spontaneously clear infection. We undertook this study to investigate the impact of alcohol on likelihood of HCV spontaneous viral clearance stratified by sex groups. Methods: Pooled data from an international collaboration of prospective observational studi...

Significance During chronic viral infection, CD8 ⁺ T cells are gradually deprived of their principal effector functions and irreversibly loose their plasticity to develop into memory populations, precluding the establishment of long-lasting protective immunity. Relevant host-derived factors directing this T-cell exhaustion process have remained elu...

Background Bayesian evolutionary analysis (coalescent analysis) based on genetic sequences has been used to describe the origins and spread of rapidly mutating RNA viruses such as Influenza, Ebola, HIV and hepatitis C virus (HCV). Methods Full length subtype 1a and 3a sequences from early HCV infections from the International Collaborative of Inci...

Figure S1. Longitudinal expression of NK cell marker and receptors on CD56dimCD16+ NK cells. Figure S2. Decreased expression of NKG2A on CD56brightCD16‐ NK cells correlates with decreased IFN‐λ3 plasma levels in individuals with CC IFN‐λ3 genotype and spontaneous resolution of acute HCV. Figure S3. IFN‐λ3 plasma levels do not correlate with NK ce...

Introduction Polymorphisms in the type III interferon IFN‐λ3 and the killer cell immunoglobulin‐like receptor (KIR) genes controlling the activity of natural killer (NK) cells can predict spontaneous resolution of acute hepatitis C virus (HCV) infection. We hypothesized that IFN‐λ3 polymorphism may modulate NK cell function during acute HCV. Metho...

CD8 T cell exhaustion is an immunosuppressive mechanism by which chronic viruses escape adaptive immune response. Yet, the direct implication of cytokines as well as the precise candidates involved in this process have remained elusive. Here we demonstrate the direct contribution of IL-2 and IL-15 on several aspects of CD8 T cell exhaustion. Using...

Symptomatic acute HCV infection and interferon lambda 4 (IFNL4) genotypes are important predictors of spontaneous viral clearance. Using data from a multicohort database (Injecting Cohorts [InC3] Collaborative), we establish an independent association between host IFNL4 genotype and symptoms of acute hepatitis C virus infection. This association po...

Interest in the role of antibody-dependent cellular cytotoxicity (ADCC) in protection from HIV infection has grown since analyses of the RV144 HIV vaccine trial results found ADCC correlated with protection. Natural killer (NK) cells are among the effector cells that mediate ADCC. The level of antibody-induced NK cell activation depends on NK cell...

The majority of acute hepatitis C virus (HCV) infection (75%) leads to chronicity and liver diseases. While 25% of infected individuals can naturally clear the infection, long­term protection is elusive and reinfection can occur. We have previously showed that protection from viral persistence upon HCV reinfection correlates with expansion of HCV s...

The majority of acute hepatitis C virus (HCV) infection (75%) leads to chronicity and liver diseases. While 25% of infected individuals can naturally clear the infection, long-term protection is elusive and reinfection can occur. We have previously showed that protection from viral persistence upon HCV reinfection correlates with expansion of HCV s...

Background: We aimed to characterize the natural history of hepatitis C virus (HCV) reinfection and spontaneous clearance following reinfection (reclearance), including predictors of HCV reclearance. Methods: Data were synthesized from the 9 prospective cohorts of the International Collaboration of Incident Human Immunodeficiency Virus and HCV i...

Background: Hepatitis C virus (HCV) testing and counselling have the potential to impact individual behaviour and transmission dynamics at the population level. Evidence of the impact of an HCV-positive status notification on injection risk reduction is limited. The objective of our study was to (1) assess drug and alcohol use and injection risk b...

Hepatitis C virus (HCV) currently infects approximately 250,000 individuals in Canada and causes more years of life lost than any other infectious disease in the country. In August 2011, new therapies were approved by Health Canada that have achieved higher response rates among those treated, but are poorly tolerated. By 2014/2015, shortcourse, wel...

Activation of hepatic stellate cells (HSCs) is a key event in the initiation of liver fibrosis, characterized by enhanced extracellular matrix production and altered degradation. Activation of HSCs can be modulated by cytokines produced by immune cells. Recent reports have implicated the proinflammatory cytokine IL-17A in liver fibrosis progression...

Background & aims: Development of a vaccine against hepatitis C virus (HCV) has been hindered by our limited understanding of immune correlates of protection during real-life exposure to the virus. We studied the immune response during HCV reinfection. Methods: We analyzed blood samples from participants in the Montreal Acute Hepatitis C Injecti...

Hepatitis C virus (HCV) infection is a major cause of morbidity and mortality in the HIV co-infected population. Interferon-alpha (IFN-α) remains a major component of anti-HCV therapy despite its deleterious effects on the immune system. Furthermore, IFN-α was recently shown to diminish the size of the latent HIV reservoir. The objectives of this s...

The majority of individuals who become acutely infected with hepatitis C virus (HCV) develop chronic infection and suffer from progressive liver damage while approximately 25% are able to eliminate the virus spontaneously. Despite the recent introduction of new direct-acting antivirals, there is still no vaccine for HCV. As a result, new infections...

Mouse mammary tumor virus superantigens (vSAGs) are notorious for defying structural characterization, and a consensus has yet to be reached regarding their ability to bridge the TCR to MHC class II (MHCII). In this study, we determined the topology of the T cell signaling complex by examining the respective relation of vSAG7 with the MHCII molecul...

Unlabelled: Although 20%-40% of persons with acute hepatitis C virus (HCV) infection demonstrate spontaneous clearance, the time course and factors associated with clearance remain poorly understood. We investigated the time to spontaneous clearance and predictors among participants with acute HCV using Cox proportional hazards analyses. Data for...