Bert Van Es
email: vanes@science.uva.nl

Shota Gugushvili
email: gugushvili@eurandom.tue.nl

Weak convergence of the supremum distance for supersmooth kernel deconvolution

Keywords: Deconvolution, kernel density estimator, Rayleigh distribution, supremum distance

We derive the asymptotic distribution of the supremum distance of the deconvolution kernel density estimator to its expectation for certain supersmooth deconvolution problems. It turns out that the asymptotics are essentially different from corresponding results for ordinary smooth deconvolution.

Introduction and results

Consider the classical deconvolution problem: let X 1 , . . . , X n be i.i.d. observations, where X i = Y i + Z i and Y i and Z i are independent. Assume that the unobservable Y i have distribution function F and density f , and that the random variables Z i have a known density k. Note that the density g of X i is equal to the convolution of f and k. The nonparametric deconvolution problem is the problem of estimating f or F from the observations X i . Thus we want to recover the distribution of Y i using the contaminated measurements X i . Additional information on measurement error models and many practical examples can be found in [START_REF] Carroll | Measurement Error in Nonlinear Models: A Modern Perspective[END_REF].

A popular density estimator for this problem is the deconvolution kernel density estimator introduced in [START_REF] Carroll | Optimal rates of convergence for deconvolving a density[END_REF] and [START_REF] Stefanski | Deconvoluting kernel density estimators[END_REF]. This estimator is defined as

f nh (x) = 1 2π ∞ -∞ e -itx φ w (ht)φ emp (t) φ k (t) dt = 1 nh n j=1 v h x -X j h , (1)
with

v h (u) = 1 2π ∞ -∞ φ w (s) φ k (s/h) e -isu ds.
Here w denotes a kernel function, h > 0 is a bandwidth, φ emp is the empirical characteristic function of the sample defined by φ emp (t) = (1/n) n j=1 e itX j , and φ w and φ k denote the characteristic functions of w and k, respectively.

Note that (1) is not a standard kernel density estimator, because the kernel function v h depends on the bandwidth h. For an introduction to the estimator (1) see e.g. [START_REF] Wand | Kernel Smoothing[END_REF].

The rate of decay to zero at minus and plus infinity of the modulus of the characteristic function φ k , and consequently the smoothness of k, is crucial

A C C E P T E D M A N U S C R I P T

ACCEPTED MANUSCRIPT

to the asymptotic behaviour of (1). Two cases have been distinguished, the ordinary smooth case, where |φ k | decays algebraically to zero, and the supersmooth case, where it decreases exponentially. The asymptotics in the ordinary smooth case are essentially the same as for a kernel estimator of a higher order derivative of a density, see e.g. [START_REF] Fan | Asymptotic normality for deconvolution kernel density estimators[END_REF], [START_REF] Fan | A note on asymptotic normality for deconvolution kernel density estimators[END_REF] and [START_REF] Van Es | Simple kernel estimators for certain nonparametric deconvolution problems[END_REF]. The asymptotics in the supersmooth case have been studied e.g. in [START_REF] Fan | Asymptotic normality for deconvolution kernel density estimators[END_REF] and van Es andUh (2004, 2005).

Notice that the above papers study local properties of the estimator (1), i.e. its pointwise behaviour. We, on the other hand, will focus on the asymptotic behaviour of the supremum distance of the estimator to its expectation, which provides a global measure of its performance. Accordingly, define

M n = sup 0≤x≤1 |f nh (x) -E [f nh (x)]|. (2)
The fact that the supermum is taken over [0, 1] is not a restriction of generality and is for convenience only. One could have considered any interval [a, b]. An alternative here is to consider the integrated squared error of the estimator f nh . This was done in [START_REF] Holzmann | Integrated square error asymptotics for supersmooth deconvolution[END_REF].

The asymptotic distribution of the supremum distance similar to (2), namely

sup x∈[0,1] (g(x)) -1/2 |g nh (x) -E [g nh (x)]|
, for an ordinary kernel density estimator g nh in the direct density estimation setting (i.e. in the error-free case) was derived in [START_REF] Bickel | On some global measures of the deviations of density function estimates[END_REF]. Owing in a certain sense to the similarity of the asymptotics in the ordinary smooth deconvolution problem to that in the direct density estimation problem, qualitatively similar results

were obtained in [START_REF] Bissantz | Non-parametric confidence bands in deconvolution density estimation[END_REF] in the ordinary smooth deconvolution problem for the supremum distance sup x∈[0,1] (g(x)

) -1/2 |f nh (x) -E [f nh (x)]|.
Normalisation with g(x) is explainable by the fact that the expression for the asymptotic variance in the asymptotic normality theorem for the estimator f nh (x) in the ordinary smooth deconvolution problem involves g(x), see [START_REF] Fan | Asymptotic normality for deconvolution kernel density estimators[END_REF]. No direct extension of the methods used in [START_REF] Bickel | On some global measures of the deviations of density function estimates[END_REF] to the supersmooth deconvolution problem is possible and derivation of the asymptotic distribution of (2) requires a different approach. This is precisely the task of the present paper. Notice that in (2) we do not have to normalise with g(x), because the asymptotic variance in the asymptotic normality theorem for this case does not depend on g, but only on the error density k (in some global way), see [START_REF] Van Es | Asymptotic normality of kernel type deconvolution estimators[END_REF].

We now state the conditions on the density k and kernel w, which will be used throughout the paper. The condition on k which defines supersmooth deconvolution is given in Condition 1.

Condition 1. Assume that

φ k (t) = C|t| λ 0 exp -|t| λ /µ (1 + o(|t| -1 )) (3)
as |t| → ∞, for a constant 0 < λ ≤ 2 and some constants µ > 0, λ 0 ∈ R and

C ∈ R. Furthermore, let φ k (t) = 0 for all t ∈ R.
Condition 1 is stronger than the usual condition on k in supersmooth deconvolution given e.g. in van Es and Uh ( 2005), where the term o(|t| -1 ) is not present and one just has the asymptotic equivalence.

Condition 2. Let φ w be real-valued, symmetric and have support

[-1, 1]. Let φ w (0) = 1, and assume φ w (1 -t) = At α + o(t α ) as t ↓ 0 for some constants A and α ≥ 0.
For examples of such kernels see for instance [START_REF] Van Es | Asymptotic normality of kernel type deconvolution estimators[END_REF].

The next theorem establishes the asymptotic distribution of M n , which could prove useful for the construction of uniform confidence bands around f . Since it will appear repeatedly in the paper, we will write ζ(h) for exp(1/(µh λ )).

Theorem 1. Assume Condition 1 for λ = 2 and Condition 2 and let E [X 2 j ] < ∞. Let V denote a positive random variable with a Rayleigh distribution with

density f V (x) = x exp[-x 2 /2]I [x≥0] . Then, as n → ∞ and h → 0, √ n h λ(1+α)+λ 0 -1 ζ(h) M n D → 1 2 √ 2 A πC µ λ 1+α Γ(α + 1) V, (4)
where Γ denotes the gamma function.

By assuming λ = 2 we restrict our selves to deconvolution problems for error distributions with characteristic functions that have an exponential tail like the characteristic function of a normal density. The most important case covered by this condition is standard normal deconvolution, where λ = 2, λ 0 = 0, µ = 2 and C = 1. The condition λ = 2 seems to be essential in the proof of Lemma 3, specifically in (8), where we prove a condition for tightness of the remainder process R (1) n . Whether it can be relaxed by other approaches, avoiding

tightness, remains open.

The rate of convergence in Theorem 1 once again reflects the difficulty of the supersmooth deconvolution problem compared to the ordinary smooth deconvolution. Furthermore, unlike in ordinary smooth deconvolution, see [START_REF] Bissantz | Non-parametric confidence bands in deconvolution density estimation[END_REF], in order to obtain the asymptotic distribution of M n , we do not have to subtract a drift term. This also has a parallel when considering the asymptotics of the ISE[f nh ] in the supersmooth deconvolution, see Holzmann and Boysen (2006) for additional details. Notice also that unlike the direct density estimation or the ordinary smooth deconvolution, see [START_REF] Bickel | On some global measures of the deviations of density function estimates[END_REF] and [START_REF] Bissantz | Non-parametric confidence bands in deconvolution density estimation[END_REF], the limit distribution in (4) is not Gumbel, which confirms the conjecture in [START_REF] Bissantz | Non-parametric confidence bands in deconvolution density estimation[END_REF] for the

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT case λ = 2.
2 Proof of Theorem 1

The proof of Theorem 1 is based on a decomposition of f nh (x) in van Es and Uh (2005), which is the basis of the proof of their asymptotic normality theorem. We have

f nh (x) = 1 πC h λ 0 -1 1 ǫ φ w (s)s -λ 0 exp(s λ /(µh λ ))ds 1 n n j=1 cos X j -x h + R (1) n (x) + R (2) n (x) + R (3) n (x), (5)
where

R (l) n (x) = (1/n) n j=1 R (1)
n,j (x), l = 1, 2, 3, and

R (1) n,j (x) = 1 C 1 π h λ 0 -1 1 ǫ cos s X j -x h -cos X j -x h × φ w (s)s -λ 0 exp(s λ /(µh λ ))ds R (2) n,j (x) = 1 2πh ǫ -ǫ exp is X j -x h φ w (s) 1 φ k (s/h) ds R (3) n,j (x) = 1 2πh -ǫ -1 + 1 ǫ exp is X j -x h φ w (s) × 1 φ k (s/h) - 1 C |s| h -λ 0 exp(|s| λ /(µh λ )) ds.
We will write R (l) n , l = 1, 2, 3 for the stochastic processes

R (l) n = (R (l) n (x)) x∈[0,1] .
Notice that these processes belong to the space C[0, 1].

Now the rough idea is to derive the asymptotic distribution of the supremum of the first summand in (5) minus its expectation and to show that the remainder terms are negligible. Define the process

U n as U n (x) = n -1/2 n j=1 U n,j (x),
where

U n,j (x) = cos((X j -x)/h) -E [cos((X j -x)/h)].
Note that this is a process with expectation equal to zero at every x. Write

S n = sup 0≤x≤1 |U n (x)|.

ACCEPTED MANUSCRIPT

Lemma 1. Under the conditions of Theorem 1 we have, as n → ∞ and h → 0,

S n D → sup 0≤x≤2π |W (x)|,
where W is a zero mean Gaussian process on [0, 2π] with covariance function

Cov(W (x 1 ), W (x 2 )) = (1/2) cos(x 1 -x 2 ).
Proof. Replacing x by yh, by the periodicity of the cosine function we have

for h ≤ (2π) -1 that S n = sup 0≤x≤1 |U n (x)| = sup 0≤x≤1 1 √ n n j=1 cos X j -x h -E cos X j -x h = sup 0≤y≤1/h 1 √ n n j=1 cos X j -yh h -E cos X j -yh h = sup 0≤y≤1/h 1 √ n n j=1 (cos(Y j -y) -E [cos(Y j -y)]) = sup 0≤y≤2π 1 √ n n j=1 (cos(Y j -y) -E [cos(Y j -y)]) , = sup 0≤y≤2π |W n (y)|,
where Y j = (X j /h) mod 2π and the process W n on [0, 2π] is given by W n (y) = n -1/2 n j=1 (W n,j (y) -E [W n,j (y)]) with W n,j (y) = cos(Y j -y). It follows that we have to study the convergence of the process W n (x) -

By

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT E [W n (x)] which belongs to C[0, 2π].
According to Prohorov's theorem and in particular Theorem 8.1 of [START_REF] Billingsley | Convergence of Probability Measures[END_REF], it suffices to show weak convergence of the finite dimensional distributions and tightness of the sequence.

By the multivariate central limit theorem in the triangular array scheme or Cramer-Wold device, see Theorem 7.7 in [START_REF] Billingsley | Convergence of Probability Measures[END_REF], the finite dimensional distributions of the process W n converge to multivariate normal distributions with covariances given by Cov(W (y 1 ), W (y 2 )) = (1/2) cos(y 1 -y 2 ). To prove tightness, we will verify conditions of Theorem 12.3 of [START_REF] Billingsley | Convergence of Probability Measures[END_REF].

First of all, notice that the sequence W n (0) is tight, because the asymptotic normality of W n (0) follows by a univariate Lyapunov central limit theorem in a trinagular array scheme, see Theorem 7.3 in [START_REF] Billingsley | Convergence of Probability Measures[END_REF]. Furthermore, for an arbitrary positive η,

P |W n (y 2 ) -E [W n (y 2 )] -(W n (y 1 ) -E [W n (y 1 )])| ≥ η ≤ 1 η 2 Var[W n,j (y 2 ) -W n,j (y 1 )] ≤ 1 η 2 E [(W n,j (y 2 ) -W n,j (y 1 )) 2 ] ≤ 1 η 2 (y 2 -y 1 ) 2 ,
which follows from the fact that

| cos(Y j -y 2 ) -cos(Y j -y 1 )| = 2 sin 2Y j -y 2 -y 1 2 sin y 1 -y 2 2 ≤ |y 1 -y 2 |.
Here we used the inequality | sin x| ≤ |x|. Therefore W n converges weakly to a zero mean Gaussian process W on [0, 2π] with covariance function Cov(W (y 1 ), W (y 2 )) =

(1/2) cos(y 1 -y 2 ). By the continuous mapping theorem, see Theorem 5.1 in [START_REF] Billingsley | Convergence of Probability Measures[END_REF], the supremum of |W n | then converges weakly to the supremum of the absolute value of the limit process, which proves the lemma.

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

Lemma 2. With V as in Theorem 1, we have

sup 0≤x≤2π |W (x)| D = 1 2 √ 2 V. ( 6
)
Proof. Let N 1 and N 2 denote two independent standard normal random variables and let us define the process W by W = ( W (x)) x∈[0,2π] , where

W (x) D = 1 2 √ 2(N 1 cos x + N 2 sin x).
Since the covariance function Cov(W (x 1 ), W (x 2 )) of the process W , given by

(1/2) cos(x 1 -x 2 ), equals Cov( W (x 1 ), W (x 2 )) by Cov 1 2 √ 2(N 1 cos x 1 + N 2 sin x 1 ), 1 2 √ 2(N 1 cos x 2 + N 2 sin x 2 ) = 1 2 (cos x 1 cos x 2 + sin x 1 sin x 2 ) = 1 2 cos(x 1 -x 2 ), it follows that W D = W . Next write 1 2 √ 2(N 1 cos x + N 2 sin x) = 1 2 √ 2 N 2 1 + N 2 2 N 1 N 2 1 + N 2 2 cos x + N 2 N 2 1 + N 2 2 sin x = 1 2 √ 2 N 2 1 + N 2 2 (cos ξ cos x + sin ξ sin x) = 1 2 √ 2 N 2 1 + N 2 2 cos(x -ξ), (7)
for a ξ such that cos

ξ = N 1 / N 2 1 + N 2 2 and sin ξ = N 2 / N 2 1 + N 2 2 .
The supremum of the absolute value of ( 7)

is equal to (1/2) √ 2 N 2 1 + N 2 2 = (1/2) √ 2V,
where V has a Rayleigh distribution. This entails (6).

Lemma 3. Let a n = √ nh -λ(1+α)-λ 0 +1 (ζ(h)) -1 denote the normalising sequence in Theorem 1. For l = 1, 2, 3 we have

a n (R (l) n -E [R (l) n ]) P → 0
as n → ∞ and h → 0. Here 0 denotes the zero process on [0, 1].

Proof. To prove the lemma, we will apply Prohorov's theorem, and in particular Theorem 8.1 of [START_REF] Billingsley | Convergence of Probability Measures[END_REF]. Firstly, notice that for a fixed x the re-

mainder terms a n (R (l) n (x)-E [R (l) n (x)]
) vanish in probability, which was proved in van [START_REF] Van Es | Asymptotic normality of kernel type deconvolution estimators[END_REF]. This implies that the finite dimensional vectors of the processes

a n (R (l) n -E [R (l) n ]
) also converge in probability to null vectors. To establish tightness, we will again verify conditions of Theorem 12.3 of [START_REF] Billingsley | Convergence of Probability Measures[END_REF]. Notice that when x = 0, the sequence

a n (R (l) n (0) -E [R (l) n (0)]) is
tight, since it converges to zero in probability. Furthermore, for an arbitrary positive η we have

P a n |R (1) n (x 2 ) -E [R (1) n (x 2 )] -(R (1) n (x 1 ) -E [R (1) n (x 1 )])| ≥ η ≤ a 2 n η 2 Var[R (1) n (x 2 ) -R (1) n (x 1 )] = a 2 n η 2 1 n Var[R (1) n,1 (x 2 ) -R (1) n,1 (x 1 )] ≤ a 2 n η 2 1 n E [(R (1)
n,1 (x 2 ) -R

(1)

n,1 (x 1 )) 2 ] ≤ a 2 n η 2 1 C 2 1 π 2 1 n h 2(λ 0 -1) (x 2 -x 1 ) 2 × K 2 1 ǫ 1 -s h 2 φ w (s)s -λ 0 exp(s λ /(µh λ ))ds 2 = K 2 a 2 n η 2 1 C 2 1 π 2 1 n h 2(λ 0 -2)-2 (x 2 -x 1 ) 2 × 1 ǫ 1 -s)φ w (s)s -λ 0 exp(s λ /(µh λ ))ds 2 = O 1 n h 2(λ 0 -2)-2+2(2+α)λ (ζ(h)) 2 a 2 n 1 η 2 (x 2 -x 1 ) 2 = O h 2(λ-2) 1 η 2 (x 2 -x 1 ) 2 (8) = O(1) 1 η 2 (x 2 -x 1 ) 2 .
where K is some constant. Here we used Lemma 5 of van Es and Uh (2005), which states that

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 1 ǫ s -λ 0 (1 -s) β φ w (s) exp(s λ /(µh λ ))ds ∼ A µ λ h λ 1+α+β ζ(h)Γ(α + β + 1), (9)
and the fact that for 0 ≤ s ≤ 1 and 0 ≤ x 1 < x 2 ≤ 1 we have cos s

X j -x 2 h -cos X j -x 2 h -cos s X j -x 1 h + cos X j -x 1 h = x 2 x 1 1 s ∂ 2 ∂u∂v cos v X j -u h -cos X j -u h dudv = x 2 x 1 1 s 1 h sin v X j -u h + v X j -u h 2 cos X j -u h dudv ≤ x 2 x 1 1 s 1 h 2 (|X j | + 1 + h) dudv ≤ 1 h 2 (|X j | + 1 + h)(1 -s)|x 1 -x 2 |.
Hence the process a

n (R (1) n -E [R (1) n ]) is tight.
In order to prove tightness of the process a n (R (2) n -E [R (2) n ]), note that, as above, for positive η

P a n |R (2) n (x 2 ) -E [R (2) n (x 2 )] -(R (2) n (x 1 ) -E [R (2) n (x 1 )])| ≥ η ≤ a 2 n η 2 1 n E [(R (2) n,1 (x 2 ) -R (2) n,1 (x 1 )) 2 ] ≤ 4 a 2 n η 2 1 4π 2 h 2 1 n ǫ -ǫ s h φ w (s) 1 φ k (s/h) ds 2 (x 2 -x 1 ) 2 = 4 a 2 n η 2 1 4π 2 h 4 1 n ǫ -ǫ sφ w (s) 1 φ k (s/h) ds 2 (x 2 -x 1 ) 2 ≤ 4a 2 n 1 4π 2 h 4 1 n (2ǫ) 2 ǫ 2 ǫ -ǫ 1 φ k (s/h) ds 2 1 η 2 (x 2 -x 1 ) 2 ≤ 4a 2 n 1 π 2 h 4 1 n ǫ 4 sup -ǫ≤s≤ǫ 1 φ k (s/h) 2 1 η 2 (x 2 -x 1 ) 2 ≤ 4a 2 n 2 C 2 π 2 1 n (ǫ/h) 4-2λ 0 exp(2(ǫ/h) λ /µ) 1 η 2 (x 2 -x 1 ) 2 = o(1) 1 η 2 (x 2 -x 1 ) 2 , A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT
where K is some constant and where we used the fact that for 0

≤ s ≤ 1, exp is X j -x 2 h -exp is X j -x 1 h ≤ cos s X j -x 2 h -cos s X j -x 1 h + sin s X j -x 2 h -sin s X j -x 1 h ≤ 2s h |x 1 -x 2 |, (10)
which follows by converting the differences of sines and cosines into products and using the fact that | sin x| ≤ |x|. Consequently, the process a n (R (2) n -

E [R (2) n ]) is tight.
To prove tightness of the process a n (R

(3) n -E [R (3) n ]
), we first introduce the function u, given by

u(y) = C|y| λ 0 exp(-|y| λ /µ) φ k (y) -1. (11)
By Condition 1 this function is bounded on R\(-δ, δ), where δ is an arbitrary positive number. Moreover, by (3) the function xu(x) is also bounded and both functions vanish at plus and minus infinity. It follows that (s/h)u(s/h) is bounded and tends to zero for all fixed s with |s| ≥ ǫ as h → 0.

Using the function u, rewrite R

(3) n,j (x) as follows

R (3) n,j (x) = 1 2πh -ǫ -1 + 1 ǫ exp is X j -x h φ w (s) × 1 φ k (s/h) - 1 C |s| h -λ 0 exp(|s| λ /(µh λ )) ds = 1 2πh -ǫ -1 + 1 ǫ exp is X j -x h φ w (s) × 1 C |s| h -λ 0
exp(|s| λ /(µh λ ))u(s/h)ds.

Next note that, as above, for positive η we have by (10) that

P a n |R (3) n (x 2 ) -E [R (3) n (x 2 )] -(R (3) n (x 1 ) -E [R (3) n (x 1 )])| ≥ η ACCEPTED MANUSCRIPT ≤ a 2 n η 2 1 n E [(R (3) n,1 (x 2 ) -R (3) n,1 (x 1 )) 2 ] ≤ a 2 n η 2 1 4π 2 h 2 1 n -ǫ -1 + 1 ǫ φ w (s) × 1 C |s| h -λ 0 exp(|s| λ /(µh λ )) s h u s h ds 2 (x 2 -x 1 ) 2 = o(1) 1 η 2 (x 2 -x 1 ) 2
and hence a n (R (3) n -E [R (3) n ]) is tight. By Prohorov's theorem each of the three processes now converges weakly to the zero process. Since the convergence in distribution to a constant entails convergence to the same constant in probability, this concludes the proof of the lemma.

Finally, we combine the obtained results to prove Theorem 1.

Proof of Theorem 1. The proof is immediate from Lemmas 1-3 just proved, the fact that by (9)

  Lemma 6 of van Es and Uh (2005) we know that Y j D → Un(0, 2π) as h → 0 for each j, where Un(0, 2π) denotes the uniform distribution on [0, 2π]. Hence by the dominated convergence theorem we get that Cov cos Y j -y 1 , cos Y j -y 2


Padmam Puneet

Mairi A Mcgrath

Kee Hwee

Tay

Lamyaa Al-Riyami

Justyna Rzepecka

Shabbir M Moochhala

Shazib Pervaiz

Margaret M Harnett

William Harnett

Alirio J Melendez

: A Melendez

Helminth product ES-62 protects against septic shock via TLR4-dependent, autophagosomal MyD88 degradation

Sepsis is one of the most challenging health problems worldwide. Toll-like receptor (TLR) 4 and TLR2 are highly upregulated in phagocytes from sepsis patients, however, shock-inducing inflammatory responses mediated by these TLRs are inhibited by ES-62, an immunomodulator secreted by the filarial nematode, Acanthocheilonema viteae. ES-62 subverts TLR4 signalling to block TLR2 and TLR4-driven inflammatory responses via autophagosomal-mediated downregulation of the TLR adaptor-transducer, MyD88. In vivo, ES-62 protects mice against endotoxic and polymicrobial septic shock using TLR4-mediated induction of autophagy and is protective even when administered after the induction of sepsis. Given that current treatments for septic shock are inadequate, ES-62's autophagy-dependent mechanism of action might form the basis of an urgently required therapeutic intervention against this life-threatening condition.

The incidence of sepsis has seen a dramatic increase over the past few decades and this is most likely due to a combination of ageing in the population, an increase in the number of invasive clinical procedures and the use of immunosuppressants [START_REF] Dombrovskiy | Rapid increase in hospitalization and mortality rates for severe sepsis in the United States: a trend analysis from 1993 to 2003[END_REF][START_REF] Padkin | Epidemiology of severe sepsis occurring in the first 24 h in intensive care units in England, Wales, and Northern Ireland[END_REF][START_REF] Vincent | The prevalence of nosocomial infection in intensive care units in Europe. Results of the European Prevalence of Infection in Intensive Care (EPIC) Study. EPIC International Advisory Committee[END_REF] . Indeed, sepsis is one of the most common causes of admission to intensive care units worldwide [START_REF] Dombrovskiy | Rapid increase in hospitalization and mortality rates for severe sepsis in the United States: a trend analysis from 1993 to 2003[END_REF][START_REF] Padkin | Epidemiology of severe sepsis occurring in the first 24 h in intensive care units in England, Wales, and Northern Ireland[END_REF][START_REF] Vincent | The prevalence of nosocomial infection in intensive care units in Europe. Results of the European Prevalence of Infection in Intensive Care (EPIC) Study. EPIC International Advisory Committee[END_REF] . In the USA alone, around three-quarters of a million cases of sepsis occur every year and despite all efforts, the fatality rate for sepsis remains between 30% and 50% (reviewed in Ref. 4). The initiation of sepsis is commonly triggered by bacterial infection. Host responses to bacterial infections are primarily mediated by the innate immune system, in particular by neutrophils and monocytes-macrophages [START_REF] Balk | Severe sepsis and septic shock. Definitions, epidemiology, and clinical manifestations[END_REF][START_REF] Alexander | Bacterial lipopolysaccharides and innate immunity[END_REF] , which express pattern recognition receptors (PRRs) that bind conserved molecular structures named pathogen-associated molecular patterns (PAMPs) that are shared by microorganisms [START_REF] Alexander | Bacterial lipopolysaccharides and innate immunity[END_REF][START_REF] Janeway | Innate immune recognition[END_REF][START_REF] Gay | Structure and function of Toll receptors and their ligands[END_REF] . Stimulation of PRR signaling pathways by PAMPS such as lipopolysaccharide (LPS) initiates secretion of inflammatory cytokines and other proinflammatory mediators which promote both the elimination of infectious agents and the induction of tissue repair [START_REF] Janeway | Innate immune recognition[END_REF][START_REF] Gay | Structure and function of Toll receptors and their ligands[END_REF][START_REF] Doyle | Toll-like receptors: from the discovery of NFkappaB to new insights into transcriptional regulations in innate immunity[END_REF][START_REF] O'neill | The family of five: TIR-domain-containing adaptors in Toll-like receptor signalling[END_REF] . However, excessive production of inflammatory mediators can lead to tissue damage and septic shock [START_REF] Parrillo | Septic shock in humans. Advances in the understanding of pathogenesis, cardiovascular dysfunction, and therapy[END_REF][START_REF] Exley | Endotoxaemia and serum tumour necrosis factor as prognostic markers in severe acute pancreatitis[END_REF][START_REF] Wang | HMG-1 as a late mediator of endotoxin lethality in mice[END_REF] . Thus, aberrant hyper-production of particular proinflammatory mediators, such as TNF, IL-1β, macrophage migration inhibitory factor (MIF) and the high mobility group-1 protein (HMGB1), associated with PAMP-mediated signalling via the PRRs Toll-like receptor 2 (TLR2) and 4 (TLR4) [START_REF] Parrillo | Septic shock in humans. Advances in the understanding of pathogenesis, cardiovascular dysfunction, and therapy[END_REF][START_REF] Exley | Endotoxaemia and serum tumour necrosis factor as prognostic markers in severe acute pancreatitis[END_REF][START_REF] Wang | HMG-1 as a late mediator of endotoxin lethality in mice[END_REF][START_REF] Bustin | At the crossroads of necrosis and apoptosis, signaling to multiple cellular targets by HMGB1[END_REF][START_REF] Andersson | High mobility group 1 protein (HMG-1) stimulates proinflammatory cytokine synthesis in human monocytes[END_REF] , results in a systemic inflammation that triggers the pathological outcomes of sepsis, including systemic vascular leakage, tissue injury, multi-organ failure and death [START_REF] Martin | The epidemiology of sepsis in the United States from 1979 through 2000[END_REF][START_REF] Balk | Severe sepsis and septic shock. Definitions, epidemiology, and clinical manifestations[END_REF][START_REF] Parrillo | Septic shock in humans. Advances in the understanding of pathogenesis, cardiovascular dysfunction, and therapy[END_REF][START_REF] Exley | Endotoxaemia and serum tumour necrosis factor as prognostic markers in severe acute pancreatitis[END_REF] .

We have shown recently that ES-62, a well-characterized, homogeneous phosphorylcholine (PC)-containing glycoprotein secreted by the filarial nematode, Acanthocheilonema viteae associates with TLR4 and subverts its proinflammatory functions in mouse macrophages, dendritic cells and mast cells [START_REF] Goodridge | Immunomodulation via novel use of TLR4 by the filarial nematode phosphorylcholine-containing secreted product, ES-62[END_REF][START_REF] Melendez | Inhibition of FcεRI-mediated mast cell responses by ES-62, a product of parasitic filarial nematodes[END_REF] . This ability of ES-62 to inhibit LPS-triggered production and secretion of pro-inflammatory mediators prompted us to investigate its therapeutic potential in septic shock caused by bacteria.

RESULTS

ES-62 blocks endotoxin-triggered signaling

TLR4 is constitutively expressed on the surface of most immune system cells and following incubation of macrophages derived from healthy humans with ES-62, the helminth product colocalised with, and bound to, TLR4 (Fig. 1a-b). Such ES-62-TLR4 complexes were internalized and surface TLR4 expression on macrophages was strongly downregulated by 2 h post-ES-62 exposure (Fig. 1c). Consistent with this, pre-incubation of macrophages with ES-62 also inhibited the LPS-mediated activation of the inflammatory gene transcription factor NF-κB (Fig. 1d), as well as the release of various proinflammatory mediators including TNF, IL-1β, IL-6, MIP-1α and HMGB1 (Fig. 1e). Although ES-62 did not lead to downregulation of cell surface expression of TLR2 at 2 h post-incubation (Fig. 1f), cells pretreated with ES-62, had significantly reduced NF-κB activation and production of proinflammatory mediators following treatment with the TLR2 agonist BLP (Fig. 1g &h). We therefore investigated the potential mechanisms by which ES-62 influenced not only TLR4, but also TLR2 signals, and found that treatment with the helminth product leads to the downregulation of the key signal transducer, MyD88 (Fig. 1i). This effect was selective because ES-62 did not similarly modulate the adaptor protein Mal (also known as TIRAP) protein expression (Supplementary Figure 1a). Moreover, this immunomodulatory action of ES-62 reflected subversion of classical TLR4 signalling because LPS did not downregulate either MyD88 or Mal under these conditions (Supplementary Figure 1a). Confocal microscopy established not only that such differential regulation of MyD88 expression similarly occurred in mouse bone marrow-derived macrophages (BMM) but also that while MyD88 expression was dispersed throughout the cells in resting and LPS-stimulated cells, the residual MyD88 expression resulting from ES-62 treatment appeared to be restricted to a perinuclear location (Supplementary Figure 1b). Quantitative laser scanning cytometric analysis confirmed that while over a period of 16 h LPS did not downregulate MyD88 (Supplementary Figures 2ac), ES-62 strongly downregulated MyD88 expression in BMM (Supplementary Figures 2d &e).

Highly upregulated TLRs in phagocytes from sepsis patients

We next analyzed the responses of infiltrating macrophages and neutrophils obtained from the peritoneal cavity, as well as from peripheral blood, of patients with sepsis. For this study we enrolled 30 patients with severe sepsis as well as 15 healthy volunteer control subjects. The major diagnoses in the patients were systemic bacterial infection in 12 (40%) patients, intraabdominal infection in 12 (40%) patients, and trauma in 6 (20%) patients. The neutrophils and macrophages from both anatomical sites of the above mentioned patients showed increased cell surface expression of both TLR4 and TLR2 when compared to macrophages and neutrophils from healthy individuals (Fig. 2a & b and results not shown). Such data were consistent with previous clinical findings, which indicated an upregulation of TLRs on neutrophils and monocytes-macrophages during the early stages of sepsis [START_REF] Mizushima | Methods in mammalian autophagy research[END_REF] . Nevertheless, and consistent with the effect of ES-62 on macrophages from healthy humans described above, the helminth product also induced downregulation of TLR4 (Fig. 2a) and MyD88 (Fig. 2c), but not TLR2 (Fig. 2b) expression in neutrophils and macrophages from sepsis patients.

Reflecting this, pretreatment of these cells with ES-62 prevented LPS-and BLP-triggered degranulation (Fig. 2d), as well as the secretion of various proinflammatory mediators (Fig. 2e). Collectively, the data in Figures 1 and2 suggest that ES-62 may block sepsis-mediated proinflammatory responses by downregulating expression of TLR4 and MyD88, the latter a key regulator of TLR responses.

Mechanism of ES-62-induced TLR4 and MyD88 downregulation

As ES-62, but not LPS, association with TLR4 appeared to lead to downregulation of TLR4 from the cell surface and consequent degradation of MyD88, we investigated whether these TLR4 ligands induced differential vesicular trafficking as a switch mechanism to trigger antiinflammatory versus proinflammatory signalling, respectively. While treatment with ES-62 induced trafficking of TLR4 and MyD88 to EEA-1-containing early endosomes within 20 min, it resulted in their localization in LAMP-1-containing late endosomes and/or lysomomes at later time-points (by 1 h). In contrast, in LPS-treated cells TLR4 and MyD88 recycled to the cell surface via the early endosomes but, importantly, did not localize to the lateendosomes and/or lysosomes even up to 4h post-stimulation (Fig. 3a & 3b and results not shown). Consistent with this, LPS induced the downregulation of TLR4 from the cell surface of macrophages at early (≤ 2h) but not late (overnight) time points and analysis of permeabilised cells confirmed that this early surface downregulation did not reflect TLR4 degradation but rather its internalization and, with time, recycling to the cell surface (Supplementary Figure 3).

We therefore hypothesised that ES-62 mediated the trafficking of TLR4 and MyD88 to lysosomes for degradation by subverting TLR4-regulation of autophagy [START_REF] Delgado | Toll-like receptors in control of immunological autophagy[END_REF] , which functions both to target intracellular proteins for degradation and as an effector of TLR-mediated innate and adaptive immune responses [START_REF] Delgado | Toll-like receptors in control of immunological autophagy[END_REF][START_REF] Deretic | Multiple regulatory effector roles of autophagy in immunity[END_REF] . Thus, to address whether ES-62 promotes sequestration and degradation of pro-inflammatory TLR signalling complexes in an autophagy-dependent manner, we investigated whether LPS and ES-62 differentially induced LC3-containing vesicles, a marker of autophagosome formation 20 . Indeed, while LPS does not induce vesicular localisation of the autophagy marker LC3 (Fig. 3c), in ES-62 treated cells there was a clear redistribution of the diffuse cytosolic expression of LC3 to a classical autophagosomepunctuate or vesicular pattern [START_REF] Klionsky | Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes[END_REF] and also colocalisation of TLR4 and MyD88 to such LC3containing vesicles (Fig. 3c).

To provide further evidence of the involvement of autophagy in ES-62-mediated degradation of MyD88, additional sets of experiments were conducted. Firstly, electron microscopy analysis over a period of 8h clearly showed various stages of autophagosome formation and then resolution in primary human macrophages exposed to ES-62 (Fig. 4a).

Additionally in primary mouse macrophages, siRNA mediated gene silencing of Atg5 or Atg7 (Fig. 4b), genes that play critical roles in autophagosome formation, virtually abrogated ES-62-induced degradation of MyD88 (Fig. 4c). Thirdly, as complete and effective autophagy is a function of an efficient autophagic flux, we investigated the effect of ES-62 on p62 (also known as the sequestrome or SQSTM1) expression as p62 binds to LC3II, the cleaved and post-translationally modified form of LC3 that increases in expression during autophagy and this p62-LC3II complex is degraded in cells undergoing efficient autophagy. Indeed, using primary mouse macrophages analysis of the kinetics of p62 expression upon exposure to ES-62 clearly indicated an early accumulation of p62 together with LC3II, followed at later time points by degradation of the two proteins (Fig. 4d). The early accumulation of p62 protein expression reflected a rapid and transient upregulation of p62 mRNA (Fig. 4e) but analysis in the presence of the protein synthesis inhibitor, cycloheximide (Fig. 4f) revealed that this upregulation masked concomitant degradation of p62 indicative of a highly dynamic and efficient autophagic flux. Finally, whereas inhibitors of lysosomal degradation (E64D and pepstatin A;

ES-62 protects mice from endotoxic shock

To determine whether ES-62 could protect against pathogen product-mediated proinflammatory responses in vivo, we examined if it reduced inflammation and lethal shock in mouse models of sepsis. In the first study, we induced LPS-associated endotoxemia in mice and lethality was monitored over time. Treatment of mice with ES-62, starting 2 h before LPS administration, resulted in a dose-dependent protection and notably, 100% of the mice survived endotoxaemia when administered ES-62 at a dose of 2 μg/animal, while none of the control mice survived (Fig. 5a and Supplementary Figure 4a). Analysis of tissue samples from the mice pretreated with ES-62 showed a significant suppression of LPS-induced pathology both in terms of the level of immune cell infiltration and damage to the lungs and liver (Fig. 5b), as well as a reduction in the serum concentrations of TNF, IL-1β, IL-6, MIP-1α and HMGB1 (Fig. 5c). The same protective effect was observed in a BLP-induced model of shock (data not shown).

ES-62 protects mice from polymicrobial sepsis

Our data showing that ES-62 is protective against bacterial product-induced shock are consistent with the well-established role of TLR4 and TLR2 in the pathogenesis of septic shock [START_REF] Salomão | TLR signaling pathway in patients with sepsis[END_REF][START_REF] Weighardt | Role of Toll-like receptor responses for sepsis pathogenesis[END_REF] . However, although an informative model, experimental endotoxic shock reproduces human sepsis only in part. This is because it does not involve the replication and dissemination of bacteria, which results in the production of pathogenic inflammation by simultaneously triggering multiple PRRs including TLR2, 4 and 9 [START_REF] Salomão | TLR signaling pathway in patients with sepsis[END_REF][START_REF] Weighardt | Role of Toll-like receptor responses for sepsis pathogenesis[END_REF] . Moreover, it was possible that ultimately, the anti-inflammatory effects of ES-62 could be detrimental to the host by impairing the capacity of the immune system to fight such bacterial infections, as observed for anti-TNF treatments [START_REF] Phillip | Mediator modulation therapy of severe sepsis and septic shock: does it work?[END_REF][START_REF] Eichacker | Risk and the efficacy of antiinflammatory agents: retrospective and confirmatory studies of sepsis[END_REF] . We therefore investigated whether ES-62 protects against septic shock due to polymicrobial peritonitis and sepsis without compromising the host's ability to fight infection, in the cecal ligation and puncture (CLP) model that resembles the scenario in human infectious sepsis [START_REF] Hubbard | Cecal ligation and puncture[END_REF] .

In a prophylactic multi-dose study, similar to that of endotoxin-induced shock, ES-62 at a dose of 2 µg/animal was sufficient to fully protect against CLP-induced death (Fig. 6a) and Supplementary Figure 4b) and multiorgan-inflammatory damage (Fig. 6b). This was accompanied by a significant lowering of peritoneal levels of neutrophils and macrophages (Fig. 6c), as well as by a reduction in serum levels of a wide range of cytokines and chemokines and also HMGB1 (Fig. 6d). Measurement of peritoneal bacteria counts revealed no significant difference between untreated and ES-62-treated mice (Fig. 6e). Interestingly, however, blood bacteria levels were significantly lower in ES-62 treated mice (Fig. 6f). These results suggest that ES-62 reduces inflammatory responses without compromising control of bacterial infection; indeed it appeared to aid control, perhaps by preventing neutropenia, as neutrophils are obligatory for effective killing of bacteria in the blood, and it has recently been suggested that an optimal concentration of neutrophils is required for the clearance of bacteria in suspension [START_REF] Li | A critical concentration of neutrophils is required for effective bacterial killing in suspension[END_REF] . Perhaps consistent with this idea that ES-62 prevents CLP-driven neutropenia, analysis of nitric oxide (NO) production revealed that while high levels of NO were found in the bronchoalveolar lavage (BAL) but not in plasma following CLP, treatment with ES-62 resulted in an inverse profile of NO production (Supplementary Figure 4c). This was in agreement with the pattern of migration of neutrophils and monocytes (Fig. 6c), cells that are likely to be the major producers of this effector, under these disease and therapeutic conditions.

ES-62 triggered autophagy mediates protection in vivo

Our data demonstrating that ES-62 prevents TLR2-and TLR4-driven pro-inflammatory responses by downregulating MyD88, together with the discovery that ES-62 protects mice from the lethal systemic inflammation triggered by multiple polymicrobial ligands, agrees with our previous in vitro studies showing that in mouse macrophages, ES-62 not only disrupts TLR4-signalling, but also inhibits TLR2-and TLR9-, but not MyD88-independent TLR3-, driven pro-inflammatory responses in a TLR4-dependent manner [START_REF] Goodridge | Immunomodulation via novel use of TLR4 by the filarial nematode phosphorylcholine-containing secreted product, ES-62[END_REF] . In order to further prove that the mechanism by which ES-62 exerts its protective functions in vivo was via such subversion of TLR4 signaling, we carried out the CLP procedure in Tlr4 -/-and Tlr2 - /-mice. Previous studies had shown that genetic deletion of either Tlr4 or Tlr2 only confers a very small degree of protection against polymicrobial sepsis, whereas deletion of MyD88 is more protective [START_REF] Weighardt | Cutting edge: myeloid differentiation factor 88 deficiency improves resistance against sepsis caused by polymicrobial infection[END_REF] . Consistent therefore with the hypothesis that ES-62 was acting to suppress signaling via multiple TLRs by triggering degradation of MyD88 via TLR4, ES-62 was unable to provide protection from CLP in Tlr4 -/-mice but it was able to confer full protection against the lethal outcome of polymicrobial sepsis in Tlr2 -/-mice (Fig. 7a). To address whether such ES-62-mediated subversion of TLR4 signaling in vivo also reflected induction of autophagy, we exploited in vivo siRNA silencing [START_REF] Puneet | SphK1 regulates proinflammatory responses associated with endotoxin and polymicrobial sepsis[END_REF][START_REF] Sethu | TNFα-induced inflammatory responses in vivo are mediated by PLD1[END_REF] to knockdown protein expression of Atg5 or Atg7 in this model (Fig. 7b). Following silencing of Atg5 or Atg7 in vivo, ES-62 was no longer able to exert protective effects against CLP (Fig. 7c). Collectively, these data clearly demonstrated that in vivo, ES-62 acts via TLR4, and similar to our in vitro data, show that it signals via an autophagosomal-dependent mechanism to subvert TLR responses (summarised in Supplementary figure 5) in order to protect mice from the lethal inflammation that is invariably fatal in experimental sepsis.

Therapeutic potential of ES-62 in polymicrobial sepsis

We next investigated whether ES-62 was protective when administered 1, 3, and 6 h after the CLP procedure. Notably, ES-62 still conferred 100% protection against polymicrobial sepsis when applied 1 h after the CLP procedure (Fig. 8a) and substantial protection was also observed when ES-62 was first administered 3 h (80% survival) or even 6 h (70% survival) after CLP (Fig. 8a). However, protection is much reduced at later time points (20% survival at 8 h) and lost completely by 12 h after CLP (results not shown). Nevertheless, it is clear that ES-62 can be highly effective even when injected after the onset of sepsis. Consistent with this, analysis of blood samples taken from control mice and animals treated with ES-62 at different time points associated with protection after CLP, showed a significant reduction in the plasma concentrations of TNF, IL-1β, IL-6, MIP-1α and HMGB1 from mice treated with the parasite product (Fig. 8b,c). Furthermore, histological examination of the lungs and liver showed a substantial reduction in inflammation and tissue damage in the CLP mice treated therapeutically with ES-62 (Fig. 8d). As injection of ES-62 into naive mice did not affect the levels of circulating leukocytes (results not shown), treatment with ES-62 is sufficient to lower systemic levels of TNF, IL-1β, IL-6, MCP-1α and HMGB1, and to reduce cellular infiltrates at the site of inflammation below levels that are lethal for the host, without causing leukopenia.

Finally, in an attempt to reproduce the clinical scenario, we utilized a broad spectrum antibiotic, as patients diagnosed as having septic shock will be immediately treated with a course of such antibiotics at a high-dose for up to three days. Although early use of antibiotics is essential in treating sepsis, such antibiotic treatment has to be applied with caution. This is especially the case since the very high doses required to control the overwhelming infection in septic shock patients, can only be administered for a short time period because of the potential for severe hepatotoxicity. Hence, novel (combination) therapies that potentially reduce this risk are desirable. Co-Amoxiclav injection at 20 mg/kg (this antibiotic and dose are currently used clinically) 6h after the CLP induction provided 20% protection from death (Fig. 8e).

However, a combination therapy of ES-62 with Co-Amoxiclav, 6h after the induction of CLP, provided 100% protection from death (Fig. 8e).

Discussion

It is well established that TLR signaling in response to overwhelming bacterial infection leads to the hyper-secretion of proinflammatory mediators associated with shock. Autophagy is a homeostatic mechanism primarily designed to promote cell survival and function and hence acts to protect against a range of pathologies including degenerative disorders and cancer [START_REF] Delgado | Toll-like receptors in control of immunological autophagy[END_REF][START_REF] Deretic | Multiple regulatory effector roles of autophagy in immunity[END_REF] .

However, there is increasing evidence that autophagy can also play multiple roles in the regulation of innate and adaptive immune responses, in particular promoting clearance of microbial infection and preventing autoimmunity [START_REF] Delgado | Toll-like receptors in control of immunological autophagy[END_REF][START_REF] Deretic | Multiple regulatory effector roles of autophagy in immunity[END_REF] . For example, with respect to microbial infection, TLR-mediated induction of autophagy has recently been implicated as an anti-microbial mechanism. This is not only in terms of elimination of pathogens by innate cells but also with respect to the delivery of cytosolic PAMPs to endosomal TLRs as well as in the development of protective adaptive immune responses both by promotion of antigen presentation and by acting as an effector of T H 1-T H 2 polarization. Indeed while T H 1-type cytokines such as IFNγ and TNF promote autophagy and clearance of bacteria, this response is suppressed by T H 2 cytokines such as IL-4 and IL-13 (reviewed in Refs. 18,19). It was therefore quite surprising that TLR-signalling (either directly by TLR ligands or culture with bacteria) was found not to be associated with autophagosome formation in primary macrophages [START_REF] Saitoh | Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1beta production[END_REF] but this has now been suggested to reflect the autophagy-suppressing effects of NF-κB activation [START_REF] Deretic | Multiple regulatory effector roles of autophagy in immunity[END_REF] . Indeed, blocking of autophagy with the inhibitor 3-methyladenine (3-MA) was demonstrated to result in hyper-induction of IL-1β in response to LPS and such aberrant production of this pro-inflammatory cytokine was also found in autophagy-defective Atg16L1-and Atg7-deficient macrophages [START_REF] Saitoh | Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1beta production[END_REF] . Moreover, although TLR ligands were found to strongly upregulate p62 (STSQM1) in a MyD88-dependent manner in primary human keratinocytes, this synthesis of p62 was predominantly required for NF-κB activation and inflammation [START_REF] Lee | Autophagy Negatively Regulates Keratinocyte Inflammatory Responses via Scaffolding Protein p62/SQSTM1[END_REF] . Thus while siRNA silencing of genes critical for autophagy, beclin-1 or Atg5, resulted in increased expression of p62, NF-κB activation and pro-inflammatory mediator release, silencing of p62 blocked NF-κB activation and production of TNF and IL-6 [START_REF] Lee | Autophagy Negatively Regulates Keratinocyte Inflammatory Responses via Scaffolding Protein p62/SQSTM1[END_REF] . These studies therefore suggest that autophagy may also act to limit TLR-mediated proinflammatory cytokine production [START_REF] Saitoh | Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1beta production[END_REF][START_REF] Lee | Autophagy Negatively Regulates Keratinocyte Inflammatory Responses via Scaffolding Protein p62/SQSTM1[END_REF] . Our finding that ES-62-mediated suppression of the hyperinflammatory responses associated with septic shock reflects triggering of Atg5 and Atg7-dependent autophagosome formation and efficient p62-LC3-mediated autophagic flux and lysosomal degradation of TLR4 and MyD88 clearly resonates with the latter proposal and suggests that manipulating autophagy induction could represent a potent anti-inflammatory strategy.

Collectively, our data indicate that ES-62 appears to act to suppress pathological proinflammatory TLR signaling by inducing autophagy to downregulate the expression of TLR4 and MyD88. In this way, ES-62 acts to reduce serum concentrations of inflammatory mediators resulting from TLR signaling to levels that do not induce shock and death but remain sufficient to promote bacteria clearance. Thus, in contrast to the situation generally observed following treatment with other anti-inflammatory agents, including nitric oxide synthase inhibitors and anti-TNF monoclonal antibodies, which increase shock lethality due to suppression of the host's ability to fight infection [START_REF] Phillip | Mediator modulation therapy of severe sepsis and septic shock: does it work?[END_REF][START_REF] Eichacker | Risk and the efficacy of antiinflammatory agents: retrospective and confirmatory studies of sepsis[END_REF] , ES-62 protects against polymicrobial peritonitis and sepsis as well as preventing multi-organ damage resulting from catastrophic inflammatory responses. The ability of ES-62 to enhance the host's capacity to clear infection might reflect inhibition of neutropenia, and relating to this, a possible contributing factor is that whilst the parasite product dampens TLR-mediated pro-inflammatory cytokine production, it leaves nitric oxide responses intact [START_REF] Goodridge | Modulation of macrophage cytokine production by ES-62, a secreted product of the filarial nematode Acanthocheilonema viteae[END_REF] . This is an intriguing finding as studies have suggested that both of these responses exhibit MyD88-dependence [START_REF] Henneke | Cellular activation, phagocytosis, and bactericidal activity against group B streptococcus involve parallel myeloid differentiation factor 88-dependent and independent signaling pathways[END_REF] . However, this apparent discrepancy may now be reconciled by our present findings that whilst ES-62 acts to downregulate MyD88, it leaves Mal levels unaffected, perhaps suggesting that TLR4-Mal interactions might be sufficient for NO-induction as has previously been described for some cellular responses including NF-κB and MAP kinase activation and some cytokine production [START_REF] Horng | TIRAP: an adapter molecule in the Toll signaling pathway[END_REF][START_REF] Horng | The adaptor molecule TIRAP provides signalling specificity for Tolllike receptors[END_REF] . Consistent with this, the Mycobacterium tuberculosis-derived lipomannan, triacyl-LM, which is an agonist of TLR2-TLR1, has been reported to promote TNF, IL-12 p40 and NO production through MyD88 and/or Mal [START_REF] Doz | Acylation determines the toll-like receptor (TLR)-dependent positive versus TLR2-, mannose receptor-, and SIGNR1-independent negative regulation of proinflammatory cytokines by mycobacterial lipomannan[END_REF] . Also of interest given the potential bactericidal role of NO, there is evidence that the Mal allele variant 180L protects against septic shock [START_REF] Ferwerda | Functional and genetic evidence that the Mal/TIRAP allele variant 180L has been selected by providing protection against septic shock[END_REF] by displaying increased TLR2,4 responses and resistance to infection. It is therefore tempting to speculate that infection with helminth that secrete products such as ES-62 that can downregulate MyD88 may have promoted selection of this allele.

Finally, ES-62 can exhibit protective effects, even when administered therapeutically several hours after the CLP procedure. In this regard, research efforts have recently been directed to finding a mechanism for limiting the excessive inflammation triggered by TLRs as a way of providing potential novel therapies to treat sepsis and other inflammatory disorders [START_REF] Daubeuf | TLR4/MD-2 monoclonal antibody therapy affords protection in experimental models of septic shock[END_REF][START_REF] Sha | Therapeutic effects of TAK-242, a novel selective Toll-like receptor 4 signal transduction inhibitor, in mouse endotoxin shock model[END_REF][START_REF] Leon | Discovery and development of toll-like receptor 4 (TLR4) antagonists: a new paradigm for treating sepsis and other diseases[END_REF] . Thus, administration of ES-62, or perhaps more likely synthetic small molecule derivatives, alone or in combinations with antibiotics, after the initiation of sepsis might offer a suitable novel therapeutic tool for the treatment of septic shock as well as other microbialmediated diseases in humans where "out of control" inflammation can often lead to fatal outcomes.

A full description of the Methods employed is found in the supplementary information. Human cells. Human neutrophils and macrophages were obtained from peripheral blood of "healthy" volunteers and from the peritoneal lavage and peripheral blood of patients diagnosed with sepsis as described previously. [START_REF] Balk | Severe sepsis and septic shock. Definitions, epidemiology, and clinical manifestations[END_REF][START_REF] Bone | Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis: members of the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference Committee[END_REF] ES-62. Highly purified, endotoxin-free ES-62 was prepared from the rodent filarial nematode Acanthocheilonema viteae and FITC-labeled ES-62 was prepared as described previously. [START_REF] Melendez | Inhibition of FcεRI-mediated mast cell responses by ES-62, a product of parasitic filarial nematodes[END_REF][START_REF] Mcinnes | A novel therapeutic approach targeting articular inflammation using the filarial nematode-derived phosphorylcholine-containing glycoprotein ES-62[END_REF] Flow cytometric analysis. Human cells were incubated or not with ES-62 (2 μg/ml) for indicated times. After staining with monoclonal antibody HTA125 (mouse IgG2a, anti-human TLR4) followed by rabbit-anti-mouse Ig-TRIPC (Invitrogen), the monoclonal T2.5 (mouse IgG1, Alexa Fluor 488 anti-mouse/human TLR2) or approriate mouse isotype controls, cells were analyzed by flow cytometry. For mouse cells, analysis of cell surface TLR4 expression was carried out using a biotin-labeled rat anti-TLR4/Md2 IgG2a mAb (MTS510; Ebioscience), and biotin-labeled rat IgG2a isotype control followed by streptavidin-conjugated APC.

daily for three consecutive days. In the therapeutic studies, ES-62, vehicle and/or Coamoxiclav were administered i.v. at the indicated doses and times post induction of CLP and then daily for three consecutive days. Viability and Inflammatory parameters and histology were analysed following sacrifice at the indicated times as previously described. [START_REF] Puneet | SphK1 regulates proinflammatory responses associated with endotoxin and polymicrobial sepsis[END_REF] Histology. The lungs and liver were dissected out and immersed in 10% formalin fixative for 1 day. Analysis of histological samples was carried out as previously described. [START_REF] Puneet | SphK1 regulates proinflammatory responses associated with endotoxin and polymicrobial sepsis[END_REF] Bacteria count. Mice were sacrificed at the indicated times after CLP and peritoneal lavage fluid and blood were collected. Bacterial load was analysed as previously described. [START_REF] Puneet | SphK1 regulates proinflammatory responses associated with endotoxin and polymicrobial sepsis[END_REF] Statistical analysis. The statistical significance amongst different experimental groups was analyzed by the unpaired Student's t test. All experiments were performed at least 3 times.

Fig. 4g )

 4g or lysosomal pH modifiers (Chloroquine or NH 4 Cl; Fig. 4h) rescued MyD88, p62 and LC3II from degradation induced by ES-62, the proteosomal inhibitor, lactacystin, had no effect on the biological activity of ES-62 (Fig. 4h, human cells left panel), providing further evidence that ES-62-mediated MyD88 degradation involves autophagosome-mediated mechanism(s). This mechanism of action by which ES-62 desensitizes TLR-MyD88 signaling was also seen in primary mouse macrophages (Fig. 4h, right).

Figure 1 .

 1 Figure 1. Effects of ES-62 on human macrophages. ES-62 and TLR4 co-localise as indicated

Figure 2 .

 2 Figure 2. Effect of ES-62 on phagocytes from sepsis patients. TLR4 (a) and TLR2 (b)

( c )

 c MyD88 levels in macrophages and neutrophils from patients with sepsis: control (None), phagocytes pretreated with ES-62 for 3 h (ES-62 (3 h)) and 6 h (ES-62 (6 h)): α-tubulin as loading control. (d) Degranulation of macrophages (β-hexosaminidase release) and neutrophils (β-glucorinidase release) from patients with sepsis following addition of PBS (None; basal levels) or LPS or BLP to untreated cells for 60 min (LPS or BLP), or to cells that had been pretreated with ES-62 for 6 h prior to the addition of LPS or BLP (LPS + ES-62 or BLP + ES-62). (e) Proinflammatory mediator release from macrophages 24 h after the addition of PBS, LPS or BLP to untreated macrophages (Basal, LPS or BLP), or to macrophages pretreated with ES-62 for 6 h prior to the addition of LPS or BLP (LPS + ES-62 or BLP + ES-62). Data in (d & e) are shown as means ± SD (n=3) where *p< 0.01 (Student's t-test) when comparing ES-62-treated with control LPS or BLP-stimulated cells. These experiments were undertaken at least three times.

Figure 3 .

 3 Figure 3. ES-62 targets TLR4 and MyD88 to early and late endosomes. Intracellular vesicle

Figure 4 .

 4 Figure 4. ES-62 induces autophagosome formation. (a) Electron micrographs (EM) of human

Figure 5 .

 5 Figure 5. ES-62 protects against endotoxic shock: (a) Survival curve for PBS-injected control

Figure 6 .

 6 Figure 6. ES-62 protects against polymicrobial sepsis. (a) Survival curve for sham-operated

Figure 7 .

 7 Figure 7. ES-62 protects via autophagy in vivo. (a) Survival curve following the CecalLigation and Puncture procedure in: TLR2 -/-mice (TLR2 -/-CLP), TLR4 -/-mice (TLR4 -/-CLP), TLR2 -/-mice injected with ES-62, starting 2 h prior to CLP (TLR2 -/-CLP + ES-62), TLR4 -/- mice injected with ES-62, starting 2 h prior to CLP (TLR4 -/-CLP + ES-62), and wild-type

Figure 8 .

 8 Figure 8. Therapeutic role for ES-62 in polymicrobial sepsis. (a) Survival curve following the

ACKNOWLEDGMENTS

The authors would like to thank the Biomedical Research Council of Singapore, the Medical Research Council UK, the Wellcome Trust, the Biotechnology and Biological Sciences Research Council UK and the American Asthma Foundation for their support.

Author Contributions

P.P., M.A.M., H.K.T., L.A-R. and J.R. performed experiments. S.M.M supplied special reagents. A.J.M conceived the study. A.J.M., M.M.H., S.P. and W.H. planned the experiments, supervised the study and wrote the paper. All authors analysed the data.

Co-immunoprecipitations and immunoblots were carried out as previously described. [START_REF] Melendez | Inhibition of FcεRI-mediated mast cell responses by ES-62, a product of parasitic filarial nematodes[END_REF] Cell lysates were prepared from control and ES-62-treated cells for measurement of MyD88 expression, effects of siRNA-gene silencing and effects of various inhibitors on ES-62 activity. Inhibitors: chloroquine (100 μM), NH 4 Cl (50 μM), lactacystin (10 μM) or E64D and pepstatin-A (both at 10 μg/ml) were from Sigma Aldrich (St. Louis, MO). Antibodies: anti-MyD88 was from Santa Cruz Biotechnology and anti-Atg5, anti-Atg7, anti-LC3 and anti-p62 from Abcam. Blots were also probed for anti-α-tubulin or anti-β-tubulin (Upstate) as loading controls. Appropriate HRP-conjugated secondary antibodies were used and bands visualized using the ECL Western Blotting Detection System (GE Healthcare).

Confocal and laser scanning microscopy. For human colocalisation studies, cells were treated with LPS or ES-62-FITC as indicated, then fixed and permeabilised, before being incubated with primary antibodies and then washed and stained with secondary antibodies.

Antibodies: mouse monoclonal anti-humanTLR4 antibody (HTA-125; BD Pharmingen), followed by rabbit-anti-mouse Ig-TRIPC (Invitrogen); primary mouse monoclonal anti-MyD88 (Santa Cruz Biotechnology), secondary rabbit-anti-mouse IgG-TRIPC (Invitrogen); primary rabbit anti-EEA-1 and anti-LAMP1 polyclonal antibodies (Santa Cruz Biotechnology), secondary goat anti-rabbit IgG-Alexa Fluor 594 (Invitrogen); primary mouse anti-LC3 monoclonal antibody (nanoTools Antikoerpertechnik GmbH & Co), secondary rabbit-anti-mouse IgG-FITC (Invitrogen). Cells were analysed using a Zeiss LSM510 confocal microscope. [START_REF] Melendez | Inhibition of FcεRI-mediated mast cell responses by ES-62, a product of parasitic filarial nematodes[END_REF] For the mouse studies, bone marrow-derived macrophages [START_REF] Balk | Severe sepsis and septic shock. Definitions, epidemiology, and clinical manifestations[END_REF] were fixed and permeabilised, then stained with a rabbit-polyclonal anti-MyD88 antibody followed by a FITC-conjugated