expression of immunological markers in...

EXPRESSION OF IMMUNOLOGICAL MARKERS IN INFLAMMATORY INFILTRATE CELLS OF LIVER

IN PATIENTS WITH HIV/HIV COINFECTION

N. Matsiyeuskaya, M.G. Zubritskiy

Grodno State Medical University,

Department of infectious diseases, Grodno, Belarus;

Grodno regional bureau of pathological anatomy, Department of pathomorphology, Grodno, Belarus

14th International Workshop on Co-infection: HIV, Hepatitis and

Liver Disease, 16-18 May 2018, Seville, Spain

Background

• HIV-infected patients currently remain one ofthe most significant HCV reservoir.

• The course of HCV infection in HIV-infectedpatients is characterized by the acceleration ofthe time of formation of liver cirrhosis (LC) to8.0-9.0 years

• The nature of the liver affection is combinedbecause the frequency of various hepatotoxicfactors is high in HIV positive patients.

Pathomorphology of liver in dead 119 HIV- infected patients in period from 2004 to 2010,

HCV-coinfection – 80,0%, AIDS – 88,2%

36,10%

27,70%6,70%

9,30%19,30%

0,90%Hepatitis of minimalactivityHepatitis of moderateactivityHepatitis of high activity

Liver cirrhosis, class А, В

Liver cirrhosis, class С

Matsiyeuskaya N.V., Prokopchik et al., 2013

Liver lymphocytes

The average human liver contains a population of approximately 1010

lymphocytes, which include lymphocyte subpopulations of the innate(NKT and NK cells) and adaptive immune systems (T and B cells),respectively.

The liver contains both T-and B-lymphocytes circulating in the blood,natural killers (NK), and minor subpopulations of lymphocytescontained in a small amount in the blood: γδ T-lymphocytes, 19% ofT-lymphocytes, negative for expression of CD4+ and CD8+, 5% of Tlymphocytes simultaneously expressing both markers.

The liver contains an extensive population of NK (up to 20%), NKT-

lymphocytes, B-1 lymphocytes, lymphoid dendritic cells. The Liver as an Immunological Organ Vito Racanelli and Barbara Rehermann

HEPATOLOGY 2006;43:S54-S62

Liver lymphocytes

• Liver pathology leads to change of liverlymphocytes composition results in liverinflammatory infiltrate (LII) formation.

Aim of study:

• to evaluate the expression of immunological markers in lymphocytes of LII in patients with HIV/HCV coinfection

Material & Methods:

Immunohistochemical studies were performed usingstreptavidin-biotin method («Dako») in formalin-fixed,paraffin embedded specimens of the liver.

Mouse monoclonal antibodies (anti-HIV p24, anti-humanCD4, anti-human CD8, anti-human HLA-DP, DQ, DR antigen,anti-human CD56, anti-human CD20, anti-human CD68) andpolyclonal rabbit anti-Herpes Simplex Virus 1 and 2 types instandard dilution (DakoCytomation) and goat antibodiesanti-human CD195 (CCR5) and anti-human CD184 (CXCR4)(AbDserotec), murine anti-hepatitis C (AbDserotec)antibodies, anti-human TNF-α (AbDserotec) were used.

A complex of secondary antibodies EnVision ( "DAKO“) withdiaminobenzidine has been used for visualization system

Material & Methods:

Evaluation of IHC results was performed on a lightmicroscope (magnification 100, 200, 400) in 6 random fieldsof view.

Localization of staining in cells was evaluated as well as thepercentage and intensity of positively stained cells.

Scoring criteria for intensity: 0 points – negative staining, 1– low intensity, 2 – moderate intensity, 3 – pronouncedintensity.

The percentage of lymphocytes in the liver inflammatoryinfiltrate expressing a particular marker was counted.

«Statistica» version 10 was used, data are presented as Meand interquartile range (IQRs).

Patients• Liver samples of 2 groups of patients:

• the 1st group - 18 patients with HIV/HCV

• the 2nd group – 15 patients with HIV-infection without HCV coinfection

0

10

20

30

40

50

60

70

80

90

100

Age (yrs) male (%) female (%) AIDS (%) ART (%) Liver cirrh

(%)*

Mild hep

(%)*

36,138,9

61,1

83,3

66,7

33,3

66,7

39,7

53,3

46,7

93,3

6,7 6,7

13,3

HIV/HCV

HCV

CD4+T-cells and CD8+T-cells in liver

CD8-T cells typically outnumber CD4-T cells in the liver.

The ratio of CD4 / CD8 T-lymphocytes in the liver of ahealthy person is 1: 3.5, while this ratio of these cells inthe blood is 2: 1, indicating a relatively higher level ofCTL in the liver compared to blood.

This is associated with the "specialized" response ofthe liver mainly to intracellular pathogens and tumorcell transformation more than to extracellular antigens

Gershwin, M.E. Immunopathogenesis and therapy of hepatitis C / M.E. Gershwin, J.M. Vierling,M.P. Manns // Liver Immunology / Hanley&Belfus, Ink. – Philadelphia, 2003. – 498 p.

The Liver as an Immunological Organ Vito Racanelli and Barbara Rehermann HEPATOLOGY2006;43:S54-S62

EXPRESSION OF CD4+ AND CD8+ IN INFLAMMATORY INFILTRATE LYMPHOCYTES OF LIVER

MarkersThe 1st group, n=18 The 2nd group, n=15

10-20%abs/%

>20%abs/% Me (IQR) 10–20%

abs/%>20%abs/% Me (IQR)

CD4+ (%) 11/61,1 7/38,9 20,0 (20,0–30,0) 12/80,0 3/20 20,0(20,0–

20,0)

CD8+ (%) 13/72,2 5/27,8 20,0 (20,0–30,0)* 15/100,0 0 20,0 (10,0–

20,0)

CD4+ / CD8+ 1 1

Boxplot by Group

Variable: CD8+

Median 25%-75% Min-Max 0 1

HCV 1/0

8

10

12

14

16

18

20

22

24

26

28

30

32

CD

8+

0 – HIV without HCV;

1 - HIV / HCV;

* – p<0,05, Mann–Whitney U Test

CD20+ liver lymphocytes

It is known that in healthy individualsthe relative content of B-lymphocytesin the liver is approximately the sameor slightly less than in blood

The HCV-infected liver may beconsidered an ectopic lymphoidorgan, in which B cells are stimulatedto proliferate and differentiate intoantibody-secreting cells withingerminal centers of intra-portallymphoid follicles

Gershwin, M.E. Immunopathogenesis and therapy of hepatitis C /M.E. Gershwin, J.M. Vierling, M.P. Manns // Liver Immunology /Hanley&Belfus, Ink. – Philadelphia, 2003. – 498 p.

The Liver as an Immunological Organ Vito Racanelli and BarbaraRehermann HEPATOLOGY 2006;43:S54-S62


1 - HIV / HCV;

Boxplot by Group

Variable: CD20


HCV 1/0

-10

0

10

20

30

40

50

60

CD

20

p>0,05, Mann–Whitney U Test

CD56 + liver lymphocytes (NK)Liver lymphocytes carrying the receptor NK

- the molecule of adhesion CD56+,represent one of the main populations ofLL, accounting for 20% in a healthy liver,while the NK content in the blood is lessthan 10%.

The main function of these cells is theresponse to viral infections and tumor celltransformation. It is believed that in LII forviral hepatitis the NK content can reach90%.

Gershwin, M.E. Immunopathogenesis and therapy of hepatitisC / M.E. Gershwin, J.M. Vierling, M.P. Manns // LiverImmunology / Hanley&Belfus, Ink. – Philadelphia, 2003. – 498p.


1 - HIV / HCV;

Boxplot by Group

Variable: CD56+


HCV 1/0

-10

0

10

20

30

40

50

60

70

CD

56

+

p>0,05, Mann–Whitney U

Test

EXPRESSION OF CD20, CD56, CD68 IN INFLAMMATORY INFILTRATE CELLS OF LIVER


10-20%abs/%

>20%abs/% Me (IQR) 10–20%


CD20+ (%) 7/38,9 11/61,1 25,0 (20,0–30,0) 6/40,0 9/60 .0 30,0 (20,0–

30,0)

CD56+ (%) 7/38,9 11/61,1 30,0 (20,0–30,0) 8/53,3 7/46,7 20,0 (20,0–

40,0)

CD68+ (%) 9/50 9/50 20,0 (10,0–30,0) 9/60,0 6/40,0 20,0 (20,0–

30,0)

p>0,05, Mann–Whitney U Test

HLA-DP DQ, DR

• Expression of the HLA-DP, DQ, DR marker isconstitutively presents on antigen presenting cells(APCs) and activated cells such as granulocytes,activated tissue fibroblasts, endotheliocytes,cytotoxic lymphocytes (CTL), which indicates theactivation of cells and their participation in theimmune response

• Khaitov, R.M. Immunology: a textbook for students of medicaluniversities / R.M. Khaitov, G.A. Ignatieva, I.G. Sidorovich. - Moscow:Medicine, 2000. - 432 p.

Natural ligands for CHR

CCR5-reseptors - CCL3 (MIP-1a -), CCL4

(MIP-1b), (macrophage inflammatory protein 1) CCL5 (RANTES)

CXCR4 - SDF-1 (stromal cell-derived factor-1)

CCL3 (MIP-1a -), CCL4(MIP-1b), CCL5 andCCR5 – participation in Th1 immunity responce (P. Corbeaua, J. Reynes, AIDS, 2009)

EXPRESSION OF HLA-DP, DQ, DR, CXCR4, CCR5 IN INFLAMMATORY INFILTRATE CELLS OF LIVER


10-20%abs/%

>20%abs/% Me (IQR) 10–20%


HLA-DP, DQ, DR (%)

10/55,6 8/44,4 20,0 (10,0–40,0)

5/33,3 10/66,7 40,0 (20,0–50,0)

CXCR4 15/83,3 3/18,8 10,0 (10,0–20,0)* 6/40,0 9/60,0 30,0 (10,0–

30,0)

CCR5 15/83,3 3/18,8 20,0 (10,0–20,0) 11/73,3 4/26,7 10,0 (10,0–

30,0)

CCR5 / CXCR4 1,0 (1,0–2,0)* 1,0 (0,5–1,0)

* - p<0,05, Mann–Whitney U Test

The 1st group - 18 patients with HIV/HCV

The 1 subgroup, n=12

Hepatitis of mild activity

The 2 subgroup, n=6

Liver cirrhosis

М.,26 yrs., AIDS, toxoplasmosis

encephalitis, HIV/HCV/IDU. Hep.

minimal activity, duration of

HIV infection- 9,5 yrs.

К.32 yrs. HIV/HCV co-infection,

alcoholism. Liver cirrhosis, duration of

HIV infection- 8,5 yrs.

EXPRESSION OF HLA-DP, DR, DR AND CD56 IN LIIC IN HIV/HCV CO-INFECTED PATIENTS WITH CHRONIC HEPATITIS AND LIVER

CIRRHOSIS

Markers

Chronic hepatitis of mild activity,

fibrosis 0-1, n=12

Liver cirrhosis, n=6

10-20%

abs/%

>20%

abs/%

Me, IQR 10-20%

abs/%

>20%

abs/%

Me, IQR

HLA-DP, DQ,

DR at LIIC9/75,0 4/33,3 15 (10-30) 1/16,7 5/83,3 45(30-50)*

CD56 at LIIC7/58,3 5/41,7 20 (20-30) 0 6/100 35 (30-50)*

*- p<0,05, Mann-Whitney UBoxplot by Group

Variable: HLA-DP, DQ, DR


LCIR

-10

0

10

20

30

40

50

60

70

HLA

-DP

, D

Q,

DR

Boxplot by Group

Variable: CD56+


LCIR

-10

0

10

20

30

40

50

60

70

CD

56

+ 0 – Chronic hepatitis of

mild activity

1 - Liver cirrhosis

EXPRESSION OF VIRAL ANTIGENES IN LIVER CELLS OF HIV/HCV CO-INFECTED PATIENTS WITH CHRONIC HEPATITIS

AND LIVER CIRRHOSIS

Markers

Chronic hepatitis of mild activity,

fibrosis 0-1, n=12

Liver cirrhosis, n=6

1 point

abs/%

>1 point

abs/%

Me, IQR 1 point

abs/%

>1 point

abs/%Me, IQR

HSV1 in HC 6/50,0 00,5 (0,0 -

1,0)0 5/83,3

2,0 (2,0-

2,0)*

HSV1 in KC 1/8,3 00,0 (0,0-

0,0)5/83,3 0

1,0(1,0-

1,0)*

HSV2 in KC0 0

0,0 (0,0–

0,0)3/50,0 0

0,5(0,0-

1,0)*

*- p<0,05, Mann-Whitney U; HC –hepatocytes, KC – Kupffer cells

Patents N., 29 yrs, HIV, HCV, tuberculosis, IDU, alcoholism. Liver

cirrhosis

High HSV 1 expression (3 balls) in

hepatocytes. ×400

Liver cirrhosis

The 1st group - 18 patients with HIV/HCV:productive HIV infection (p24+) of HC and KC – 8 pts vs. non

productive HIV (p24-) - 10 pts.

p24 HIV+ in HC

p24 HIV+ in KCPolychromatic staining with azure II

electron microscopy

KC

A

B

C

D

The 1st group - 18 patients with HIV/HCV:expression of immunological markers in hepatocytes (HC) and Kupffer

cells (KC) in patients with productive HIV infection (p24+) of HC and KC vs. non productive HIV (p24-)

Маркер

HIV/HCV, n=8 HIV/HCV, n=10

productive HIV infection (p24+) of

HC and KC

non productive HIV infection (p24-) of

HC and KC

1 points,

abs/%

>1points,

abs/%

Me,

IQRs

1points,

abs/%

>1 points,

abs/%

Me,

IQRs

NS4 HCV

in HC

4/50,0 3/37,5 1,0 (1,0 -

2,0) *2/20 0 0 (0,0 - 0,0)

HSV1 in

HC

5/62,0 2/25 1,0 (1,0 -

1,0) *3/30 1/10 0 (0,0 - 0,1)

HLA-DP,

DQ, DR+ in

KC

3/37,5 2/25 0,5 (0,0 -

1,0)*

1/10 0 0 (0,0 -0,0)

TNF-α in

KC

6/75,0 0 1 (0,5 -

1,0)*

1/10 0 0 (0,0 -0,0)

*- p<0,05, Mann-Whitney U

Conclusions

HIV/HCV coinfection is associated with more activatedcellular immune response and more pronouncedinflammatory reactions in the liver inflammatory infiltratecells in compare with HIV-infected patients without HCV.

Liver cirrhosis in HIV/HCV coinfection associates with moreexpressed activation of cellular immunity and emergence ofopportunistic infections with intrahepatic expression.

The productive HIV infection of hepatocytes and KC led tomore pronounced activation of KC and increasedexpression of TNF-α in them, higher expression of NS4 andHSV1 in hepatocytes.

Acknowledgments

Grodno State medical university, Belarus

• Prof. V.A. Snezhitskiy, D.Sci (Med) corresponding member of NAS of Belarus

• Prof. V.M. Tsyrcunov, D.Sci (Med)

• Prof. V.P. Andreev, MD. PHD

• Senior researcher R.I. Kravchuk, MD. PHD

Grodno regional pathological anatomical bureau

• Associated prof. N.I. Prokopchik, MD. PHD

Gomel regional pathological anatomical bureau

• V.N. Tischenko - pathomorphologist

THANK YOU!

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