The role of the CD200-CD200R pathway in human sepsis-associated immunoparalysis (SIMPA study)

Sepsis is a major medical challenge with high morbidity and mortality rates.
Many non-survivors from sepsis die in the later course of sepsis in a state of
functional failure of cellular immunity or so called immunoparalysis (or
immunosuppression or anergy). It has been proposed that this sepsis associated
immunoparalysis contributes to the enhanced susceptibility to nosocomial
infection and late mortality of septic patients that survive the first acute
phase of sepsis. It can be considered as a marker for the immune depression
that commonly follows the acute hyperinflammatory phase of sepsis.
The diminished responsiveness of blood cells involves monocytes, granulocytes
and lymphocytes. Monocyte reactivity has been investigated in cultures of
isolated cells and in whole blood assays. In both systems, the capacity to
produce tumour necrosis factor (TNF), interleukin (IL-1) and other
proinflammatory mediators was found to be markedly reduced in patients with
sepsis. Interestingly, the production of IL-1 receptor antagonist (IL-1 ra) by
monocytes was not attenuated during Gram-negative infection, suggesting that
*immunoparalysis* is not a general downregulation of cytokine production but a
purposeful adaptation of the host in favour of an anti-inflammatory phenotype.
Granulocytes isolated from patients with sepsis demonstrated a similarly
reduced responsiveness upon restimulation with bacterial antigens, as reflected
by a diminished release of IL-1β and IL-8. Also lymphocytes have been found to
secrete less proinflammatory cytokines. Furthermore, strongly reduced IFN-*
production and responsiveness have been reported in experimentally induced
immunoparalysis after endotoxin administration to healthy humans.

Although the mechanisms that underlie immunoparalysis have not been elucidated
completely, it is conceivable that anti-inflammatory cytokines, particularly
IL-10 and transforming growth factor (TGF)-β, are involved. Indeed, plasma from
patients with sepsis markedly diminished the capacity of normal monocytes to
secrete TNF, and IL-10 is a major denominator of this immunosuppressive effect
in *septic* plasma. TGF-β was found to be the causative agent of the attenuated
splenocyte reactivity in and animal model of sepsis. Other mediators released
during the initial hyperinflammatory phase of sepsis that may contribute to the
subsequent hyporesponsiveness of blood leukocytes include catecholamines,
glucocorticoids and prostaglandines.

It has been proposed that *immunoparalysis* may contribute to the enhanced
susceptibility to nosocomial infections and late mortality of patients who
survive the initial acute phase of sepsis syndrome. As a consequence,
strategies aiming to restore immune function have been developed and in part
tested in patients with sepsis. Cytokines that were able to reverse monocyte
deactivation in vitro and in animals are interferon-γ (IFN-y) and granulocyte
macrophage colony stimulating factor (GM-CSF). One pilot study was conducted
with recombinant IFN-γ in patients with sepsis and evidence for
*immunoparalysis* defined as the presence of < 30% human leukocyte antigen -
antigen d related (HLA-DR) positive monocytes for at least two days. Nine
patients were treated with daily subcutaneous injection of IFN-γ until > 50% of
their monocytes were HLA-DR positive for three consecutive days. IFN-γ
treatment restored the TNF production capacity of monocytes and was not
associated with adverse effects. Although in this small uncontrolled study the
efficacy of IFN-γ could not be determined, it was encouraging that eight
patients recovered from sepsis shortly after treatment. In a small controlled
study involving 60 infants with neutropenia and clinical signs of sepsis, daily
subcutaneous injection of recombinant human GM-CSF for seven consecutive days
was associated with an increase in neutrophil counts and a reduced mortality (3
of 30 in the GM-CSF group versus 9 of 30 in the control group).

The CD200 receptor (CD200R) is an inhibitory receptor that contributes to
control the immune response. CD200R is expressed on myeloid cells such as
monocytes, macrophages, granulocytes, as well as on lymphocytes. In myeloid
cells, it has been shown that binding of CD200 to CD200R induces CD200R
intracellular signaling leading to inhibition of inflammation via a unique
inhibitory pathway involving a direct interaction with the adaptor protein
downstream of tyrosine kinase 2 and the subsequent recruitment and activation
of Ras GTPase-activating protein (20). Splenocytes from CD200R knockout (KO)
mice show diminished LPS induced TNF-α and IL-12 supernatant levels compared to
those of wild-type mice (21). Also, addition of CD200Fc did not suppress TNF-α
and IL-12 supernatant concentrations of LPS stimulated splenocytes from the KO
mice, in contrast to those of the wild-type mice. Furthermore, in in vivo
experiments, blockade of this immune inhibitory ligand-receptor pair
(CD200/CD200R) - either pharmacologically or genetically - leads to an enhanced
susceptibility to chronic (sterile) inflammatory diseases (e.g.
collagen-induced arthritis, autoimmune encephalomyelitis) (22-25), suggesting
an immunosuppressive effect of this receptor pair pathway in vivo. In a
Gram-negative (meningitis) infection model, CD200 protects the host by limiting
inflammation (26). In that study, mice lacking CD200 have an increased
mortality associated with more inflammation, compared to wild-type mice.
Interestingly, Van der Vlist et al. from our (LTI) research group found that
the signal transduction machinery of CD200R can switch from inhibitory to
stimulatory signaling depending on the environment (submitted). Presently, the
role of CD200-CD200R pathway in the phenomenon of sepsis associated
immunoparalysis is unknown. We here hypothesize that the CD200-CD200R pathway
underlies sepsis induced immunodepression by inhibiting the responsiveness of
leukocytes.

Aim of the study:
To investigate the role of the CD200/CD200R axis on the responsiveness of whole
blood, peripheral blood mononuclear cells (PBMCs) and neutrophils to a
proinflammatory challenge in the immunoparalysed state in sepsis patients.

Primary objective:
To determine the effect of the inhibition or stimulation of the CD200/CD200R
axis on the hyporesponsiveness of whole blood, PBMCs and neutrophils to a
proinflammatory challenge in the immunoparalysed state in patients with sepsis
(with other words: can we restore the hyporesponsiveness by targeting the
CD200/CD200R axis?)

Secondary objectives:
- To establish the extent of CD200(R) expression on the surface of monocytes,
lymphocytes and neutrophils during immunoparalysis and to investigate the
underlying mechanisms.
- To evaluate whether CD200 (R) expression is correlated with disease severity
(Sequential sepsis-related Organ Failure Assessment; SOFA score) and whether
there is a difference in expression levels between survivors and
non-survivors.

Observational study

The burden from blood sampling from a line thas has already been inserted is
minimally invasive and has a neglectable risk for damage/deleterious effects
for the study subjects.