The document discusses how oxygen tension modulates the cytokine response of oral epithelium to periodontal bacteria. It finds that under different oxygen tensions, there are changes in NF-kB activation and pro-inflammatory cytokine production in response to nine species of oral bacteria in oral epithelial cells. Specifically, it shows higher NF-kB activation and cytokine production under low oxygen tensions similar to periodontal pockets compared to normal oxygen tensions. This demonstrates how the low oxygen environment in periodontal pockets can influence the inflammatory response of oral epithelium to the bacteria that cause periodontitis.

1. 5

2. Journal club
Presented by:
Satendra sharma

3. • TITLE:- Oxygen tension modulates the cytokine response of oral
epithelium to periodontal bacteria.
SOURCE:- J Clin Periodontol 2010; 37: 1039–1048.
• AUTHOR:= Grant MM, Kolamunne RT, Lock FE, Matthews
JB, Chapple ILC, Griffiths HR.

4. •Introduction
•Key words
•Aim
•Materials & methods
•Result
•Discussion
•Conclusion

5. INTRODUCTION
Periodontitis
Bacteria subgingival plaque

6. Reactive oxygen species
PROTEOLYTIC ENZYMES
oral epithelial
tissues

7. TOLL LIKE RECEPTORS
nuclear factor kappaB (NF-kB)
pro-inflammatory cytokines and chemokines
NF-kB plays a central role.

8. • The production of inflammatory cytokines/ chemokines by crevicular
epithelium in response to subgingival plaque bacteria via the TLR/NF-
kB route, promoting the local influx and activation of neutrophils, is
an important initial event in periodontal inflammation, as well as
providing a chronic host-derived stimulus.

9. inverse relationship between probing pocket depth and pocket oxygen
tension.
• Physiologically, tissues can experience between 4% and
15% oxygen; however, the gingival tissues can also
experience 21%, atmospheric oxygen conditions. At the
base of untreated periodontal pockets, the oxygen
tension can reach hypoxic levels at 2% oxygen.
• Under these conditions, the hypoxia inducible factor
(HIF)-1a is the main transcription factor that is
activated and is involved in adapting cell functions to
cope with reduced oxygen availability (Burke et al.
2003).

10. Recent data about oral epithelial cells under normal culture
conditions (21% oxygen) .

11. • Thus, this study aimed to determine epithelial
NF-kB activation and pro-inflammatory
cytokine production in response to nine
species of oral bacteria under different oxygen
tensions.
The bacterial species investigated included known periodontal
pathogens, mainly falling within the orange and red complexes , as well
as two species associated with health (Streptococcus mitis) or sites
responding well to therapy (Actionmyces viscosus).

12. CYTOKINES
An intermediate mechanism that lies between
bacterial stimulation and tissue destruction is
the production of cytokines, which stimulates
inflammatory events that activate effector
mechanisms.

13. Cytokines are defined as small soluble proteins
produced by a cell that alters the behavior or
properties of another cells locally or systemically.
Cytokines are regulatory proteins produced by
immune cells and other cells of the body.
low molecular weight polypeptides usually less
than 80 KDA .

14. Classification
• Thecytokinescanbe broadly splitinto 2 groups
Those involved in inflammatory and immune reactions.
Those involved in tissue repair and growth.
Thefirstgroupisfurthersubdividedinto:
The interleukins, which transmit information between leukocytes.
The chemokines involve in cell recruitment .
The interferons that influence lymphocyte activity.
Accordingto their origin
Those secreted by lymphocytes as lymphokines
Those secreted by monocytes/macrophages were called as monokines

15. Mechanism of Action
• Various cells secrete cytokines in response to injury or stimulation .
• Most cytokines are multifuinctional molecules that act locally and have
a target of effector cells .
• Themechanism bywhichcytokines acton targetcells
– AUTOCRINE: A mechanism involving the synthesis of a cytokine by
one cell that influences the growth and functional activities of the
cells producing the cytokine via a specific receptor on its surface.
– INTRACRINE: A mechanism involving the direct action of a cytokine
within a cell. Here the cytokine /receptor complex is rapidly
internalised by the cells and translocated to the nucleus with out
being degraded.

16. • JUXTRACRINE: a mechanism involving specific cell to cell
contact .it occurs via an interaction of a membrane bound
form of a cytokine that is normally secreted with a receptor
on an adjacent cell.
• PARACRINE: a mechanism involving the synthesis , of a
cytokine by one cell that influences the growth and
functional activities of a near by cells.

18. Cells Producing Cytokines
• MONOCYTES/MACROPHAGES
• LYMPHOCYTES(T-CELLS and B- CELLS)
• GINGIVALFIBROBLAST
• GINGIVALEPITHELIAL CELLS
• ENDOTHELIAL cells

19. Interleukin-8
INTERLEUKIN-8 (IL-8) IS A CHEMOATTRACTANT cytokine
produced by a variety of tissue and blood cells.
• Unlike many other cytokines, it has a distinct target
specificity for the neutrophil.
• The response of neutrophils to IL-8 is characterized
by migration of the cells, the release of granule
enzymes, and other intra- and extracellular changes.
• Connective tissue constituents are efficiently
degraded by neutrophil enzymes, released upon
activation.

20. BIOLOGICAL ACTIVITY
• IL-8 attracts and activates neutrophils.
• IL-8 is responsible for inducing the adhesion of neutrophils to
endothelial cells ,shape change and transmigration.
• IL-8 plays an important role in maintenance of local host –
parasite equilibrium and in the limitation of neutrophil –
associated tissue destruction (TONETTI ET AL 1998)
• AN uncontrolled release of Il-8 and resultant hyperactivity of
neutrophils can cause tissue destruction (Yoshimura etal1997).
• IL-8 can cause the release of mmp-8 by neutrophils (TONETTI ET
AL 1998)

21. Tumour Necrosis Factor Alfa & Beta
• Tumour necrosis factor ALFA and BETA are
proinflammatory cytokines that are produced mainly
by macrophages and lymphocytes respectively.
• Tumour necrosis factor beta stimulates bone resorption.
• Tumour necrosis factor has been reported to have
similar biologic effects as interleukin-1 and in
interleukin-6.
• Tumour necrosis factor is 100 to 1000 fold less potent
of bone resportion than interleukin-1.

22. • Tumour necrosis factor has been detected in
GCF of patient with gingivitis and
periodontitis.
• Tumour necrosis factor stimulated bone
resorption is inhibited by both interferon-
gamma and dexamethasone.
• Tumour necrosis factor bone resorbing
effects is potentiated by parathyroid
hormone.

23. OXYGEN

24. O2 – Chemistry, Physics & Distribution
O
Colorless,
Odorless,
tasteless &
water soluble
Param-
agnetic
Non-
metal
3rd most
abundant
element
(21% of
earth’s
atmosphere)
Two thirds of
the mass of
the human
body
Allotropes
O2
O3
O4

25. • Oxygen is a component in many of the molecules in the
human body.
• Cellular processes require oxygen to burn the calories from
food.
• Eliminate toxins and waste.
• Toxic to many anaerobic microorganisms, …....
6

26. Coefficient of utilization=
O2 taken up by tissue per 100ml
of blood / O2 content
If the delivery of O2 to
the tissues begins to fall
pO2 of tissues begins
to fall
Cellular death
7

27.  Oxidative burst:
• hydrogen peroxide,
• ROS,
• Peroxidase,
• lysozyme and hydrolytic
enzymes.
12

28. (Glickman et al.
1949)
Significant changes in gingival O2 utilization
accompany inflammatory and healing states of
gingival tissues.
(Box 1928)
The insufflations of O2 into the periodontium
appears to exert a favorable therapeutic
influence on periodontal disease.
(Takahara, 1952)
A genetically transmitted disorder of
oxidative metabolism, acatalasemia in which
severe periodontal destruction results.
Measured endogenous respiration of gingiva
might also be derived in part from O2
utilization by gingival tissue PMNs.

29. • It is value that has wide biochemical significance and according to which
it is possible to judge the character and intensity of metabolic processes
taking place in the tissues.
Normal O2 consumption of gingiva - 1.6 + 0.37 (Glickman, Turskey and Hill
1949)
Resulting values of O2 consumption are mostly expressed as
metabolic quotient QO2 = The amount of O2 in micrograms
consumed by 1 miligram of dry weight of tissues per hour (µmoles
O2/mg dry wt/hr).

30. • Endogenous respiration occurs primarily within the epithelial layer,
and connective tissue elements are only slightly active.
• QO2 of the separated epithelial layer of human gingiva ranged from
4.8-7.5 compared to 1.47 observed in subepithelial layer.
• Thus epithelial layer is the chief site of oxidative metabolism within
the gingiva. (Glickman et al. 1949, Schrader et al. 1957, Person 1961, Eichel &
Shahrik 1964)
O2 consumption in Alveolar mucosa
Epithelium > Epithelium & Connective tissue > connective tissue alone.
(Bergquist et al 1970)

31. Age
 Endogenous respiration of gingiva declines with age from a QO2
of 1.64 (0-20 years ) to 1.26 (41-60 years ) (air . 37.5°C). Volpe and
Manhold (1962)

32. Gender
 Females present a lower rate of gingival tissue respiration than
males. (Volpe et al. 1962)
 Benedict et al. (1938) observed that the differences between the
sexes markedly increased as age advances, however, no
significant difference was observed by others.
Race
 Negroes showed higher O2 consumption rates than Caucasians.
(Volpe et al. 1962)

33. Pathologic
temp.
Human gingiva pathology
QO2 (air)
(µmoles O2/mg dry
wt/hr)
Reference
37.5° C
Normal 1.60 ± 0.37 Glickman et al
(1949)
Inflamed 1.8-2.16
Slightly inflamed 1.77
Schrader et al
(1957)
Moderately inflamed 2.69
Severely inflamed 3.19-4.50
Normal 1.47
Manhold &
Volpe (1963)
Light-medium inflamed 1.79
Moderate-heavy inflamed 0.93
Slightly inflamed crevicular gingiva 5.67-7.5
Morgan &
Wingo (1966)
Moderately inflamed epithelium 6.0-7.5
Inflamed epithelium 6.25

34. • Endogenous O2 utilization by gingiva increases during mild to moderate
inflammation but decreases in sever form.
• In presence of gingival inflammation and proliferation, the oxygen
consumption of gingiva is increased, this increase may result from an
enhanced aerobic glycolysis.
• Marked increase in glycolytic pathway enzymes and specifically
cytochrome oxidase, the rate limiting enzyme.

35. The defense mechanisms of PMN cells are strongly dependent upon
certain of their oxidative metabolic processes
Deeper Pockets
Lower oxygen tension
Affect the bactericidal action of the phagocytic cells.
The gingival tissue oxidative metabolism, may also be of
importance in the pathogenesis, development and therapy
of periodontal disease.

36. O2 tension – Pocket depth & Microflora
Pocket depth
O2 tension (mean pO2)
mm of Hg
Predominant
microorganism
Moderate pockets
(5-6 mm)
15.2
Actinomyces naeslundii,
Streptococcus mutans
Deep pockets
(7-10 mm)
12
Spirochetes, Bacteroides
Intermedius,
Capnocytophaga sp,
F. Nucleatum
(Loesche et al. 1983)

37. Hyperglycemia
Glycosylation of basement membrane proteins
Thickening of basement membrane
Altered structural and physical properties of BM
Disruption of collagen fibers in BM, swelling of endothelium
Impedes oxygen diffusion

38. • Endogenous oxygen utilization and glucose oxidation in the
gingiva of are significantly higher in the nondiabetic as compared
with the diabetic individual.

39. I always think of leaving smoking but
to think that I need a cigarette.

40. • Nicotine is quickly absorbed and starts release of catecolamines
which causes peripheral vascular constriction and decrease
perfusion rate up to 42% and in turn O2 supply to tissues.
• CO in cigarette smoke reduce O2 content of blood.
• Arterial O2 saturation of Hb is lower in smokers than non smokers.
(Tirlapur et al 1983)

41. • Smoking distinctly decreases tissue blood flow, oxygen tension,
and aerobic metabolism independent of smoking status.
(Sorensen, 2008)
• Smokers exhibit possibly lower function of oxygen efficiency in
healthy gingiva and reduced ability to adapt the function in
inflamed gingiva than non-smokers due to impairment in gingival
microcirculation in both healthy and inflamed gingiva. (Hanioka et
al. 2000)

42. Long or continued emotional stress
causes a constant adrenaline induced
constriction of the blood vessels
Decreased supply of O2 and nutrient
materials for the periodontal tissues.
Lower ability of the tissues under
stress to utilize oxygen.

44. Radiation above 1.8 – 2 Gy for more
than 2 weeks
Damage to endothelial cells
(Direct & Indirect)
Hypoxia
Cell death

46. • Oxygen rich environment enhances the function of leukocytes,
activating or supporting the body defense mechanism in areas
that are already frequently poorly perfused, which in turns speeds
up the process.
• An oxygen rich milieu always inhibits the growth of anaerobic
microorganisms, effectively supporting antibiotic and surgical
therapy.
• Also inhibits any oncogenic transformations in a cell.

47. • Regenerative process are also influenced by the increase in oxygen
tension. Tissue capillarity clearly increases, and fibroblast
replication is enhanced.
• In addition, the healing process in damaged bone, as in
periodontitis, can be accelerated by oxygen therapy or even made
possible in the first place.

48. HYPOXIA INDUCIBLE FACTOR-1
• Changing oxygen levels can result in activation or
repression of certain homeostatic regulatory
genes, allowing for the survival of tissues and
cells despite fluctuating environmental
conditions.
• Genes such as HIF-1, whose activation is
prompted by hypoxic conditions, can interact
with enzymes and other transcription factors in
order to control vascularization and tissue
growth.

49. HIF-1 DOMAINSTRUCTURE
• HIF-1 is a heterodimeric transcription factor
consisting of a constitutively expressed β-
subunit and an oxygen-regulated α-subunit.
• In the α-subunit, there is an oxygen-
dependent degradation (ODD) domain, which
is hydroxylated by proline-hydroxylase- 2
(PHD-2), rendering the α-subunit vulnerable
to proteasomal degradation under normoxic
cellular conditions.

50. HIF-1 REGULATES OXYGEN HOMEOSTASIS
• HIF-1 is a major regulator of oxygen
homeostasis within cells. As a transcription
factor, it affects and regulates the expression
of dozens of genes involved in maintaining
homeostasis as oxygen concentrations change.
• HIF-1 further mediates cellular responses to
hypoxia by regulating glucose uptake and
anaerobic respiration in oxygen depleted
environments .

51. Transcriptional Regulation Controls
Angiogenesis in Hypoxia
• One important HIF-1 function is to promote angiogenesis;
HIF-1 directs migration of mature endothelial cells toward a
hypoxic environment. This is done via HIF-1 regulation of
vascular endothelial growth factor (VEGF) transcription.
• During hypoxia, HIF-1 binds the regulatory region of the
VEGF gene, inducing its transcription and initiating its
expression.
• Such endothelial cells ultimately help to form new blood
vessels, supplying the given area with oxygenated blood .

52. HIF-1 Regulates Shift to
Anaerobic Metabolism
• HIF-1 also can regulate anaerobic metabolism. When
oxygen is available, most cells produce ATP via oxidative
phosphorylation. However, in hypoxic environments, there
is a shift to anaerobic metabolism for cellular energy
production.
• HIF-1 is among the principal genes to coordinate this shift,
by inducing a variety of glycolytic enzymes and glucose
transporters such as aldolase A and pyruvate kinase M,
which help cells efficiently produce energy in hypoxic
environments.
• In addition to increasing the expression of these enzymes,
HIF-1 decreases mitochondrial oxygen consumption by
activating pyruvate dehydrogenase kinase I and halting the
citric acid cycle.

53. CANCER, INFLAMMATION,
AND HYPOXIA
• Environments surrounding metastasizing
tumor masses are often hypoxic.
• HIF-1 is a crucial protein in such masses; it
enables tumor progression by inducing
alternative metabolic pathways within cancer
cells.

54. Overexpression of HIF-1 Causes
Apoptosis
• A multitude of studies currently are being
conducted on the role of HIF-1 in hypoxia-
induced apoptosis of various cell types.
For example, Krick et al. recently reported that
overexpression of HIF-1 in alveolar epithelial
cells resulted in increased apoptosis.
• Although the exact pathways and mechanisms
involved in this process remain unclear .

57. Nf-Kb introduction
• NF-κB (nuclear factor kappa-light-chain-enhancer of
activated B cells) is a protein complex that
controls transcription of DNA.
• In most cells, NF-B complexes are inactive, residing primarily
in the cytoplasm in a complex with any of the family of
inhibitory proteins. When the pathway is activated, the
inhibitory protein is degraded and the NF-B complex enters
the nucleus to modulate target gene expression.
• These factors are involved in the control of a large number of
normal cellular and organism processes, such as immune and
inflammatory responses, developmental processes, cellular
growth, and apoptosis.

58. Nf-Kb’s structure
• In mammals, the NF-B family is composed of five related transcription
factors: p50, p52, RelA (aka p65), c-Rel and RelB.
• These transcription factors are related through an N-terminal DNA-
binding/dimerization domain, called the Rel homology domain, through
which they can form homodimers and heterodimers, which bind to a
variety of related target DNA sequences called B sites to modulate gene
expression. Rel/Nf-Kb
RelA, c-Rel and RelB contain C-
terminal transcription activation
domains (TADs), which enable
them to activate target gene
expression.
p50 and p52 do not contain C-
terminal trans activation domains;
therefore, p50 and p52
homodimers can repress
transcription.

61. AIM
To investigate the effect of different oxygen tensions upon nuclear factor-
kappa B (NF-kB) activation and the inflammatory cytokine response of
oral epithelial cells when exposed to nine species of oral bacteria.

62. MATERIAL AND METHODS

63. BACTERIA
Bacteria were harvested and washed with
sterile distilled water and heat killed for
10min. At 100C. The optical density at 600nm
of each stock suspension was measured and
used to calculate the approximate numbers of
bacteria present for subsequent multiplicity
of infection (MOI) calculations.

64. CELL CULTURE
• H400 oral epithelial cells were routinely cultured
in DMEM/F12 supplemented with 10% foetal calf
serum and glutamine under normoxic (21%)
conditions.
• This cell line was derived from an oral squamous
cell carcinoma, is adherent and shows a typical
polygonal epithelial cell morphology with
desmosomal junctions in culture.

65. • For experimentation, cells were plated at a
density of 2*10/ml and allowed to adhere
overnight under normoxic conditions before
challenge.
• To obtain media at the corresponding
atmospheric oxygen percentage as the
experimental conditions, media was pre-
equilibrated overnight in the appropriate
conditions (2%, 10% or 21% oxygen)

67. Bacterial challenge

68. RESULT

69. • Incubation of media in 2%, 10% or 21%
oxygen atmosphere over 24 h resulted in
media with dissolved oxygen concentrations
of 3.0 +- 0.5, 3.5 +- 0.5 and 4.0 +- 0.5 mg/ml at
37*C, as determined by the Winkler test.

70. • Although large amounts of IL-8 were produced
by unstimulated cells (mean SEM: 1003 +-489
pg/ml), production was generally increased
when the cells were stimulated with bacteria
or LPS.
• There was a trend among all bacterial
treatments for greater quantities of IL-8 to be
produced at 2% oxygen tension over 10%,
which was in turn 21% .

72. • The nine species of bacteria could be
subdivided into three groups according to
their ability to induce IL-8 production.
• T. forsythensis, P. gingivalis and P. intermedia
all induced high levels of IL-8 .

73. • TNF-a, produced in small amounts by
unstimulated cells, was induced by P.
intermedia, T. forsythensis P. gingivalis
showing the largest and most consistent
effect.
• P. intermedia also showed the trend for
greater cytokine production at 2% oxygen
tension over 10%, which was 21%.

75. • Immunohistochemical analysis of NF-kB
translocation indicated that this was affected
by both bacterial stimulation and oxygen
tension.
• In particular, T. forsythensis, P. gingivalis and
P. intermedia stimulated NF-kB translocation
more than any of the other bacterial species
examined.

77. DISCUSSION
• The epithelium functions as the first line of
defence against oral bacteria, providing both a
physical and responsive barrier.
• The response of the epithelium is to produce
antimicrobial factors (e.g. human b-defensin-2)
and to alert the innate and adaptive immune
systems through the production of pro-
inflammatory and immunomodulatory molecules
(e.g. IL-1a and IL-1b, IL-8 and TNF-a), after
activation of transcription factors such as NF-Kb)

78. • In this study, we have investigated the effect of a
range of oral bacteria, including examples of both
red and orange complex organisms (Socransky et
al. 1998), on the cytokine and NF-kB response of
an oral epithelial cell line and how this is
modulated by oxygen tension.
• These data demonstrated that oral epithelial cells
consistently show a greater response to T.
forsythensis, P. gingivalis and P. intermedia as
measured by nuclear translocation and activation
of NF-kB and production of IL-8 and TNF-a.

79. • Thus, two red complex bacteria, T. forsythensis
and P. gingivalis and the orange complex
bacterium P. intermedia induced the greatest
proinflammatory epithelial response at all oxygen
concentrations tested.
• have the capacity to indirectly promote
periodontal inflammation via the epithelium and
contribute to connective tissue destruction and
increased pocket depth, even under lower oxygen
conditions.

80. • The most pathogenic complex (red complex)
comprises P. gingivalis, T. forsythensis and
Treponema denticola (not tested in these
studies) and taken together, these three
organisms alongside P. Intermedia induced a
substantially greater cytokine response,
irrespective of oxygen tension.

81. CONCLUSION
• These data demonstrate a greater pro-
inflammatory host response and cell signalling
response to bacteria present in more
anaerobic conditions, and hypersensitivity of
epithelial cells to pro-inflammatory stimuli at
2% oxygen, which may have implications for
disease pathogenesis and/or therapy.

82. Thank you
LOVE LIKE OXYGEN