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Pamela Valva
Ricardo Gutierrez Children's Hospital: El Hospital de Ninos Ricardo Gutierrez
Corresponding Author
ORCiD: https://orcid.org/0000-0003-3994-2401
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: Chronic hepatitis C pathogenesis is not defined yet, so immune cell populations and cytokines in liver and peripheral blood (PB) were evaluated to elucidate their role in liver disease.
B, CTL, Th, Treg, Th1, Th17 and NK cells localization and frequency were evaluated on liver biopsies by immunohistochemistry, while frequency, differentiation, and functional status on PB by flow cytometry. TNF−α, IL−23, IFN−γ, IL−1β, IL−6, IL−8, IL−17A, IL−21, IL−10 and TGF−β expression were quantified in fresh liver biopsy by RT-qPCR and in plasma by CBA/ELISA.
Results: Liver CTL and Th1 at lobular area inversely correlated with viral load (r=−0.469, p=0.003 and r=−0.384, p=0.040). Treg correlated with CTL and Th1 at lobular area (r=0.784, p<0.0001; r=0.436, p=0.013). Th17 correlated with hepatic IL-8 (r=0.52, p<0.05) and both were higher in advanced fibrosis cases (Th17 p=0.0312, IL-8 p=0.009). Hepatic cytokines were higher in severe hepatitis cases (IL−1β p=0.026, IL−23 p=0.031, IL−8 p=0.002, TGF−β p=0.037). Peripheral NK (p=0.008) and NK Dim (p=0.018) were diminished while NK Bright (p=0.025) were elevated in patients vs donors. Naïve Th (p=0.011) and CTL (p=0.0007) were decreased, while activated Th (p=0.0007) and CTL (p=0.0003) were increased. IFN−γ production and degranulation activity in NK and CTL were normal. Peripheral cytokines showed an altered profile, particularly elevated IL−6 (p=0.008) and TGF−β (p=0.041).
Conclusions: HCV-specific and non-specific hepatic CTLs favored damage. Treg could not prevent fibrogenesis triggered by Th17 and IL-8. Peripheral T-lymphocyte differentiation stages shift, elevated cytokines levels and NK-cell count decrease would contribute to global disease.
Keywords
Immunopathogenesis, HCV, chronic liver disease, inflammatory liver infiltrate, Treg, Th17
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This is a list of supplementary files associated with this preprint. Click to download.
- RiosetalADDITIONALTABLE1.docx
ADDITIONAL TABLE 1 Clinical, biochemical, virological and histological patient features.
- RiosetalADDITIONALTABLE2.docx
ADDITIONAL TABLE 2 qRT-PCR primers description and annealing temperature.
- RiosetalADDITIONALTABLE3.xlsx
ADDITIONAL TABLE 3 Dataset analyzed during the current study
- RiosetalAdditionalFigures.pdf
ADDITIONAL FIG 1 PANEL A Infected hepatocytes in liver biopsies of CHC patients. NS3+ hepatocyte related to hepatitis (a) and fibrosis (b). PANEL B Frequency and distribution of the immune cells within liver infiltrate. Portal-periportal (a) and lobular (b) cell population frequencies. PANEL C Inflammatory activity related to total portal-periportal lymphocyte and fibrosis severity. Correlation between inflammatory activity and total porta-periportal lymphocytes (a). Inflammatory activity in relation to fibrosis severity (b). PANEL D Relationship between portal-periportal CTL and Th17 frequency and biochemical parameters of liver damage. Correlations between aspartate transaminase (AST) or alanine transaminase (ALT) levels and CTL frequency (a and b) and Th17 frequency (c). PANEL E Relationship between liver infection and the expression of IL−10 and IL−21. Correlation between infected hepatocytes (NS3+) and the hepatic expression levels of IL−10 (a) and IL−21 (b). FC: fold change. When it corresponds, the results are depicted in box plots. Horizontal lines within boxes indicate medians. Horizontal lines outside the boxes represent the 5 and 95 percentiles. Mean is indicated as +. MM: minimal – mild, MS: moderate – severe hepatitis. Advanced fibrosis (F≥3) according to METAVIR. ADDITIONAL FIG 2 PANEL A Peripheral cell populations profile in patients and donors. Percentage (upper) and absolute (lower) values of peripheral cell populations in donors (a, c) and patients (b, d). PANEL B Peripheral cell populations related to liver damage. Percentage values related to hepatitis (a, b) and fibrosis (c and d) severity. MM: minimal – mild, MS: moderate – severe hepatitis. Advanced fibrosis (F≥3) according to METAVIR. The results are depicted in box plots. Horizontal lines within boxes indicate medians. Horizontal lines outside the boxes represent the 5 and 95 percentiles. Mean is indicated as +. ADDITIONAL FIG 3 NK functionality related to liver damage. PANEL A IFN−γ production capacity of NK cells related to hepatitis (a, b) and fibrosis (c, d) severity. Percentage values (a, c) and intensity of expression (b, d). PANEL B Degranulation activity of NK cells related to hepatitis (a-f) and fibrosis (g-l). CD107a expression in total NK cells (left), NK Dim (middle) and NK Bright (right). Percentage values (a-c, g-i) and intensity of expression (d-f, j-l). MM: minimal – mild, MS: moderate – severe hepatitis. Advanced fibrosis (F≥3) according to METAVIR. The results are depicted in box plots. Horizontal lines within boxes indicate medians. Horizontal lines outside the boxes represent the 5 and 95 percentiles. Mean is indicated as +. Note: the analysis of CD107a expression absolute values yielded results similar to the percentage values, but it is not shown to simplify their visualization. ADDITIONAL FIG 4 PANEL A CTLs functionality related to liver damage. IFN−γ production capacity of CTLs related to hepatitis (a, b) and fibrosis (c, d) severity. Percentage values (a, c) and intensity of expression (b, d). PANEL B Degranulation activity of CTLs related to hepatitis (a, b) and fibrosis (c, d). Percentage values (a, c) and intensity of expression (b, d). MM: minimal – mild, MS: moderate – severe hepatitis. Advanced fibrosis (F≥3) according to METAVIR. The results are depicted in box plots. Horizontal lines within boxes indicate medians. Horizontal lines outside the boxes represent the 5 and 95 percentiles. Mean is indicated as +. Note: the analysis of CD107a expression absolute values displayed similar results to the percentage values, but it is not shown to simplify their visualization. ADDITIONAL FIG 5 Schematic representation of the results.
- RiosetalAdditionalMaterial.pdf
ADDITIONAL MATERIAL. Gating strategies for flow cytometry analysis corresponding to the peripheral lymphocyte populations quantification, T lymphocyte differentiation and functional characterization of CTLs and NK cells.
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A new preprint design is coming!
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This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research
Pamela Valva
Ricardo Gutierrez Children's Hospital: El Hospital de Ninos Ricardo Gutierrez
Corresponding Author
ORCiD: https://orcid.org/0000-0003-3994-2401
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
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History
CURRENT STATUS: Posted
Version 1
Posted 11 May, 2021
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
General Cell Biology & Physiology
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: Chronic hepatitis C pathogenesis is not defined yet, so immune cell populations and cytokines in liver and peripheral blood (PB) were evaluated to elucidate their role in liver disease.
B, CTL, Th, Treg, Th1, Th17 and NK cells localization and frequency were evaluated on liver biopsies by immunohistochemistry, while frequency, differentiation, and functional status on PB by flow cytometry. TNF−α, IL−23, IFN−γ, IL−1β, IL−6, IL−8, IL−17A, IL−21, IL−10 and TGF−β expression were quantified in fresh liver biopsy by RT-qPCR and in plasma by CBA/ELISA.
Results: Liver CTL and Th1 at lobular area inversely correlated with viral load (r=−0.469, p=0.003 and r=−0.384, p=0.040). Treg correlated with CTL and Th1 at lobular area (r=0.784, p<0.0001; r=0.436, p=0.013). Th17 correlated with hepatic IL-8 (r=0.52, p<0.05) and both were higher in advanced fibrosis cases (Th17 p=0.0312, IL-8 p=0.009). Hepatic cytokines were higher in severe hepatitis cases (IL−1β p=0.026, IL−23 p=0.031, IL−8 p=0.002, TGF−β p=0.037). Peripheral NK (p=0.008) and NK Dim (p=0.018) were diminished while NK Bright (p=0.025) were elevated in patients vs donors. Naïve Th (p=0.011) and CTL (p=0.0007) were decreased, while activated Th (p=0.0007) and CTL (p=0.0003) were increased. IFN−γ production and degranulation activity in NK and CTL were normal. Peripheral cytokines showed an altered profile, particularly elevated IL−6 (p=0.008) and TGF−β (p=0.041).
Conclusions: HCV-specific and non-specific hepatic CTLs favored damage. Treg could not prevent fibrogenesis triggered by Th17 and IL-8. Peripheral T-lymphocyte differentiation stages shift, elevated cytokines levels and NK-cell count decrease would contribute to global disease.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
This is a list of supplementary files associated with this preprint. Click to download.
- RiosetalADDITIONALTABLE1.docx
ADDITIONAL TABLE 1 Clinical, biochemical, virological and histological patient features.
- RiosetalADDITIONALTABLE2.docx
ADDITIONAL TABLE 2 qRT-PCR primers description and annealing temperature.
- RiosetalADDITIONALTABLE3.xlsx
ADDITIONAL TABLE 3 Dataset analyzed during the current study
- RiosetalAdditionalFigures.pdf
ADDITIONAL FIG 1 PANEL A Infected hepatocytes in liver biopsies of CHC patients. NS3+ hepatocyte related to hepatitis (a) and fibrosis (b). PANEL B Frequency and distribution of the immune cells within liver infiltrate. Portal-periportal (a) and lobular (b) cell population frequencies. PANEL C Inflammatory activity related to total portal-periportal lymphocyte and fibrosis severity. Correlation between inflammatory activity and total porta-periportal lymphocytes (a). Inflammatory activity in relation to fibrosis severity (b). PANEL D Relationship between portal-periportal CTL and Th17 frequency and biochemical parameters of liver damage. Correlations between aspartate transaminase (AST) or alanine transaminase (ALT) levels and CTL frequency (a and b) and Th17 frequency (c). PANEL E Relationship between liver infection and the expression of IL−10 and IL−21. Correlation between infected hepatocytes (NS3+) and the hepatic expression levels of IL−10 (a) and IL−21 (b). FC: fold change. When it corresponds, the results are depicted in box plots. Horizontal lines within boxes indicate medians. Horizontal lines outside the boxes represent the 5 and 95 percentiles. Mean is indicated as +. MM: minimal – mild, MS: moderate – severe hepatitis. Advanced fibrosis (F≥3) according to METAVIR. ADDITIONAL FIG 2 PANEL A Peripheral cell populations profile in patients and donors. Percentage (upper) and absolute (lower) values of peripheral cell populations in donors (a, c) and patients (b, d). PANEL B Peripheral cell populations related to liver damage. Percentage values related to hepatitis (a, b) and fibrosis (c and d) severity. MM: minimal – mild, MS: moderate – severe hepatitis. Advanced fibrosis (F≥3) according to METAVIR. The results are depicted in box plots. Horizontal lines within boxes indicate medians. Horizontal lines outside the boxes represent the 5 and 95 percentiles. Mean is indicated as +. ADDITIONAL FIG 3 NK functionality related to liver damage. PANEL A IFN−γ production capacity of NK cells related to hepatitis (a, b) and fibrosis (c, d) severity. Percentage values (a, c) and intensity of expression (b, d). PANEL B Degranulation activity of NK cells related to hepatitis (a-f) and fibrosis (g-l). CD107a expression in total NK cells (left), NK Dim (middle) and NK Bright (right). Percentage values (a-c, g-i) and intensity of expression (d-f, j-l). MM: minimal – mild, MS: moderate – severe hepatitis. Advanced fibrosis (F≥3) according to METAVIR. The results are depicted in box plots. Horizontal lines within boxes indicate medians. Horizontal lines outside the boxes represent the 5 and 95 percentiles. Mean is indicated as +. Note: the analysis of CD107a expression absolute values yielded results similar to the percentage values, but it is not shown to simplify their visualization. ADDITIONAL FIG 4 PANEL A CTLs functionality related to liver damage. IFN−γ production capacity of CTLs related to hepatitis (a, b) and fibrosis (c, d) severity. Percentage values (a, c) and intensity of expression (b, d). PANEL B Degranulation activity of CTLs related to hepatitis (a, b) and fibrosis (c, d). Percentage values (a, c) and intensity of expression (b, d). MM: minimal – mild, MS: moderate – severe hepatitis. Advanced fibrosis (F≥3) according to METAVIR. The results are depicted in box plots. Horizontal lines within boxes indicate medians. Horizontal lines outside the boxes represent the 5 and 95 percentiles. Mean is indicated as +. Note: the analysis of CD107a expression absolute values displayed similar results to the percentage values, but it is not shown to simplify their visualization. ADDITIONAL FIG 5 Schematic representation of the results.
- RiosetalAdditionalMaterial.pdf
ADDITIONAL MATERIAL. Gating strategies for flow cytometry analysis corresponding to the peripheral lymphocyte populations quantification, T lymphocyte differentiation and functional characterization of CTLs and NK cells.
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