Kamal Hammani

Bertrand Kibler

Julien Fatome

Sonia Boscolo

Goëry Genty

John M Dudley

Guy Millot

Christophe Finot
email: christophe.finot@u-bourgogne.fr

Goery Genty

Nonlinear spectral shaping and optical rogue events in fiber-based systems

Keywords: Fiber optics, supercontinuum, pulse shaping, extreme statistics, optical solitons

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction

Nonlinearity in single-mode optical fibers has often been regarded as a detrimental effect for the transmission of pulsed signals. However, when properly managed, nonlinearity offers great opportunity to reshape the optical spectrum of coherent light and expand its bandwidth to several hundreds of nanometers. For example, in the early 1990's, supercontinuum (SC) generated in optical fibers has found several practical applications in dense wavelength-division multiplexed communications [START_REF] Morioka | Pulse-width tunable, self-frequency conversion of short optical pulses over 200nm based on supercontinuum generation[END_REF]. The advent of photonic crystal fibers (PCF) in the late 90's has considerably widened the field of applications: by means of accurate control of the periodic arrangement of air holes and silica rods, stronger confinement of the optical field can be achieved so that the nonlinear response is enhanced by more than an order of magnitude compared to conventional fibers. PCFs also offer the crucial advantage of enabling precise engineering of the dispersive properties so that a zero dispersion wavelength (ZDW) well below the ZDW of bulk silica (1.3 µm) has become technologically feasible (indeed now commonplace). This has paved the way for efficient nonlinear processes at the wavelengths of Ytterbium technologies (1.06 µm) and within the tuning range of Ti-Sapphire lasers [START_REF] Ranka | Visible continuum generation in air-silica microstructure optical fibers with anomalous dispersion at 800 cm[END_REF]. With optical spectra routinely spanning an octave, SCs have found many disruptive applications and have stimulated numerous breakthroughs in optical coherence tomography [START_REF] Hartl | Ultrahigh-resolution optical coherence tomography using continuum generation in an air-silica microstructure optical fiber[END_REF], metrology and spectroscopy [START_REF] Holzwarth | Optical Frequency Synthesizer for Precision Spectroscopy[END_REF], biophotonics, and other fields of science.

The physics of the continuum generation is generally complex and crucially depends on the dispersion properties at the pump wavelength. However, chromatic dispersion is not the only parameter affecting the output spectrum, the input pulse duration, peak power and pulse shape also strongly influence the output and further increase the richness and diversity of the nonlinear propagation dynamics. In this paper, we choose to focus on how these parameters influence the stability characteristics of SC spectra and how they can be controlled in different regimes. In this respect, the performance of normally dispersive fibers is seen as superior to that of anomalousdispersion fibers, where large shot-to-shot fluctuations are seen both in the temporal and spectral domains [START_REF] Nakazawa | Coherence degradation in the process of supercontinuum generation in an optical fiber[END_REF]. In this context, the observation of rare but intense spikes of light [START_REF] Solli | Optical rogue waves[END_REF] in the anomalous-dispersion regime has initiated the field of optical rogue waves and has provided a new perspective for the analysis of SC stability. This, in turn, has highlighted or confirmed the crucial role of nonlinear coherent structures such as fundamental solitons [7; 8], breathers [9; 10] and rational solitons such as the Peregrine soliton [START_REF] Peregrine | Water waves, nonlinear Schrödinger equations and their solutions[END_REF].

Several review articles and books are now available to understand the physical mechanisms driving the continuum formation and its modeling using the nonlinear Schrödinger equation (NLSE) in its extended form [12; 13; 14; 15; 16]. Therefore it is not our intention here to present an additional comprehensive or analytical description of the nonlinear dynamics of pulses propagating in an optical fiber, but rather to provide an overview of our recent numerical and experimental contributions to the field of SC generation in both the normal and anomalous dispersion regimes. To this end, we restrict ourselves to fibers with a constant longitudinal dispersion profile and to the long pulse regime (i.e. picosecond pumping or longer).

Significantly, the results presented in this paper are mostly obtained at telecommunication wavelengths and with commercially available components and fibers.

In the first part of this paper, we emphasize how the normal dispersion regime is particularly well-suited for nonlinear pulse shaping, with the output pulse shape being directly controlled by adjustment of the initial amplitude and phase [START_REF] Boscolo | Nonlinear pulse shaping in fibres for pulse generation and optical processing[END_REF]. In the second part, we show that in the anomalous dispersion regime, soliton dynamics govern the temporal and spectral features of the generated SC resulting in large shot-to-shot fluctuations and associated with the emergence of extreme events. We then discuss a possible experimental solution to efficiently harness the observed fluctuations. This particular setup further allows us to experimentally generate a specific solution of NLSE which is considered as a prototype of freak events known as the Peregrine soliton.

Highly coherent supercontinuum and nonlinear spectral shaping in normally dispersive fibers

Due to the Kerr nonlinearity of silica, the propagation of intense ultrashort pulses in optical fibers is strongly affected by self-phase modulation (SPM) [START_REF] Stolen | Self-phase modulation in silica optical fibers[END_REF]. The resulting nonlinear phase directly depends on the temporal gradient of the pulse intensity profile and leads to broadening of the optical spectrum. For an ideal dispersionless fiber, this broadening would linearly increase with the propagation distance and the temporal intensity profile would remain unchanged.

However, in a real fiber, the impact of chromatic dispersion cannot be ignored, especially when the spectral bandwidth becomes substantial, and taking dispersion into account leads to radically different nonlinear dynamics depending on the dispersion regime.

In this section we consider propagation in the regime of normal dispersion. In this case, the chirp induced by the dispersion combines with the nonlinear chirp from SPM, in such as way as to enhance the temporal broadening. As a consequence, the pulse peak power progressively drops and the spectral broadening saturates [19; 20]. During propagation, the temporal intensity profile also experiences strong reshaping as illustrated in Fig. 1(a2) where an initial hyperbolic secant pulse becomes gradually close to a parabolic shape before undergoing wave-breaking phenomenon [START_REF] Anderson | Wave-breaking in nonlinear optical fibers[END_REF], and subsequently evolving into a trapezoidal-like pulse profile. The stage of wave-breaking occurs when the portions of the pulse with higher instantaneous frequencies temporally overlap with pulse portions having much lower instantaneous frequencies [20; 21; 22]. In the spectral domain, wave-breaking manifests itself as the generation of new frequencies and the appearance of sidelobes in the spectrum as seen both in the numerical simulations of Fig. 1(a1) and experimental results shown in Fig. 2(a). [START_REF] Morioka | Pulse-width tunable, self-frequency conversion of short optical pulses over 200nm based on supercontinuum generation[END_REF] show the longitudinal evolution of the spectral intensity profile, and subplots (2) show the temporal intensity at different stages of propagation. The initial pulses have a full-width at half maximum (FWHM) duration of 3 ps with an input peak power of 12 W and propagate in a highly non-linear fiber with a non-linear coefficient of 10 /W/km and a dispersion coefficient of -0.5 ps/nm/km. The results are obtained by numerical integration of the standard NLSE.

As the nonlinear dynamics involved here depends crucially on the input pulse features, it is possible to nonlinearly shape the output spectrum through accurate control of the input pulse temporal intensity or/and phase profiles. It is worth noting that this type of approach can be flexibly applied to generate spectral bandwidth exceeding significantly than that of the input pump pulse. This is obviously not possible with conventional linear shaping techniques where one cannot shift energy outside the initial pulse bandwidth [START_REF] Boscolo | Nonlinear pulse shaping in fibres for pulse generation and optical processing[END_REF]. For example, the use of initial parabolic-shaped pulses allows to prevent optical wave-breaking [23; 24] (see Fig. 1(b) and 2(b) for experimental evidence) as well as to generate a highly flat continuum with enhanced spectral energy density. The use of an initial triangular temporal profile on the other hand leads to the symmetric splitting of the pulse spectrum into two peaks of equal magnitude [START_REF] Verscheure | Pulse doubling and wavelength conversion through triangular nonlinear pulse reshaping[END_REF] (Figs. 1(c) and 2(c)), which can be advantageous for the realization of optical functions such as frequency conversion [START_REF] Parmigiani | An efficient wavelength converter exploiting a grating based saw-tooth pulse shaper[END_REF] or pulse doubling [START_REF] Latkin | Doubling of optical signals using triangular pulses[END_REF]. The input temporal phase profile also has a strong impact on the pulse evolution in the fiber and, as such, allows the generation of more advanced pulse shapes. For example, an initial Gaussian pulse with a parabolic phase profile (linear chirp) will passively reshape itself into a triangular intensity profile at a given stage of the propagation [25; 28]. Another striking example demonstrating the impact of the input phase profile is the process Experimental results of the propagation of a hyperbolic secant pulse in a dispersion-shifted fiber, of a parabolic pulse in a highly nonlinear fiber followed by spectral slicing (the different channels are plotted), of a triangular pulse and of a Gaussian pulse with a parabolic phase (subplots a, b, c and d respectively). Details of the different experimental configurations can be found in [START_REF] Finot | Beneficial impact of wave-breaking on coherent continuum formation in normally dispersive nonlinear fibers[END_REF], [START_REF] Parmigiani | Ultra-flat SPM-broadened spectra in a highly nonlinear fiber using parabolic pulses formed in a fiber Bragg grating[END_REF], [START_REF] Verscheure | Pulse doubling and wavelength conversion through triangular nonlinear pulse reshaping[END_REF] and [START_REF] Fatome | All-fiber spectral compression of picosecond pulses at telecommunication wavelength enhanced by amplitude shaping[END_REF] respectively.

Normally dispersive fibers are undoubtedly advantageous compared to anomalous fibers as far as the coherence of the generated continuum is concerned. Specifically, nonlinear propagation in the normal dispersion regime inhibits modulation instability (MI) phenomena to occur, which reduces significantly spontaneous amplification of noise and thereby shot-to-shot fluctuations.

Consequently, a high degree of coherence is maintained as illustrated in Fig. 3, where we plot the results of a few hundreds of numerical simulations with distinct input noise seeds. Such a high level of stability is associated with a single temporal coherent structure that is clearly attractive for all-optical pulse processing in highly nonlinear fibers [START_REF] Boscolo | Nonlinear pulse shaping in fibres for pulse generation and optical processing[END_REF] or the generation of SC covering the entire C-band of optical telecommunications. Indeed, several studies have reported that such continua can be sliced into several tens of high-quality high-repetition-rate picosecond channels where isolated spectral lines of the 10-GHz or 40-GHz resulting comb provide several hundreds of continuous waves [16; 23; 33]. The high degree of coherence of the generated spectra further enables efficient pulse compression with very high compression ratio as the nonlinear chirp induced by SPM can be compensated to a large extent by an optical fiber providing anomalous dispersion [26; 34; 35; 36] or by a pair of diffraction gratings [34; 37; 38]. propagating in a 500-m-long highly nonlinear fiber (nonlinear coefficient 10 /W/km, dispersion -0.5 ps/nm/km, dispersion slope 0.01 ps 2 /nm/km at 1550 nm). The results are obtained by numerical integration of the NLSE with third-order dispersion, including the quantum noise as well as 1% fluctuations on the input pump. The different shots are plotted in grey color and the average curve is plotted with a solid black line. Grey and black curves nearly coincide. This technique has been efficiently exploited in PCFs where dispersion characteristics can be accurately controlled so as to obtain a fiber with normal dispersion at all wavelengths [START_REF] Bale | Effects of fourth-order fiber dispersion on ultrashort parabolic optical pulses in the normal dispersion regime[END_REF] that enables the generation of highly broadband coherent spectra spanning more than several hundreds of nanometers. Such spectra are particularly attractive from the perspective of pulse compression down to duration as short as a few optical cycles after compensation of the nonlinear phase [40; 41].

Finally, we would like to point out that the benefits of the regime of normal dispersion in terms of nonlinear pulse shaping can be further enhanced by the additional presence of optical gain. Indeed, regardless of its initial properties, any pulse in this case tends asymptotically towards a parabolic intensity profile both in the temporal and spectral domains, combined with a highly linear chirp. The formed pulse in the amplifier then propagates in a self similar manner, experiencing an exponential increase of its amplitude, duration and spectral bandwidth. Selfsimilar parabolic pulses not only can tolerate strong nonlinearity without wave breaking, but can also be compressed to shorter pulse of very high quality [36; 42]. This makes parabolic pulse amplification an attractive alternative to chirped pulse amplification techniques at moderate energies and is beneficial for generation of high quality continua [43; 44].

Supercontinuum stability in the anomalous dispersion regime and extremes events

In this section, we review the main properties of SC generated in the anomalous dispersion regime of a nonlinear fiber.

Supercontinuum stability

The stability of the continuum obtained in the normal regime of dispersion is generally not preserved in the anomalous dispersion region (in fact the stability breaks in the vicinity of the ZDW regardless of the dispersion sign). This is because in the anomalous regime, the SC properties change significantly from shot to shot both in the temporal and spectral domains. The spectral phase fluctuations have been the subject of intense investigations as early as 2000 [START_REF] Corwin | Fundamental noise limitations to supercontinuum generation in microstructure fibers[END_REF].

But it is only at the end of the year 2007 that an article published by Solli et al has reported the experimental observation of significant fluctuations in the temporal domain [START_REF] Solli | Optical rogue waves[END_REF]. By spectrally filtering the most red-shifted part of the SC spectrum generated in a PCF pumped in the regime of anomalous dispersion, Solli et al have analyzed the statistical fluctuation of the corresponding filtered temporal intensity and found that a highly-skewed distribution that strongly differs from the Gaussian shape. By analogy with the typical L-shaped distribution observed for extreme events, the intense and extremely rare outliers of the distribution were named "optical rogue waves". Since then, the subject has become the focus of intense international research in the optical community by the large number of studies published in the last three years. This enthusiasm has indeed largely surpassed the confines of SC generation in PCFs and optical rogue waves have been reported in various nonlinear systems including filamentation [START_REF] Kasparian | Optical rogue wave statistics in laser filamentation[END_REF], optical amplifiers driven by incoherent pumps [47; 48], generation of SC in non-silica materials [START_REF] Majus | Rogue-wave-like statistics in ultrafast white-light continuum generation in sapphire[END_REF], telecommunications transmission systems [START_REF] Vergeles | Optical rogue waves in telecommunication data streams[END_REF] or optical feedback systems [51; 52; 53; 54].

An illustration of the temporal and spectral instability of the continuum generated in the anomalous dispersion region is shown in Fig. 4, which shows the results of NLSE numerical simulations similar to those in Fig. 3, but with anomalous dispersion. The nonlinear pulse dynamics is dominated by an initial stage of MI followed by soliton dynamics with splitting of the ultrashort coherent structure due to third-order dispersion. We note that similar splitting would be produced by the intra-pulse Raman effect. Pulse splitting results in the emergence of several ultrashort solitons with significant shot-to-shot amplitude and timing jitters (Fig. 4(a)) which prevents this type of SC to be sliced in multiple channels for WDM applications. The spectral intensity profile (Fig. 4(b)) is significantly broader than those typically obtained in fibers with normal dispersion but also suffers from large fluctuations. propagating in a 500-m highly nonlinear fiber (nonlinear coefficient 10 /W/km, dispersion 0.5 ps/nm/km, dispersion slope 0.01 ps/nm/km at 1550 nm). Results are obtained by the numerical integration of the standard NLSE with third-order dispersion and include the quantum noise as well as 1% of the fluctuations on the input pump. The different shots are plotted in grey and the average curve is plotted with a solid black line.

In order to illustrate quantitatively the fluctuations inherent to anomalous dispersion pumping, we present in Fig. 5 results of numerical simulations that take into account the full dispersive and nonlinear responses of the fiber through the integration of the generalized NLSE. Specifically, we show that independently of the fiber characteristics (fiber with one or two ZDWs) [55; 56] large fluctuations are present on the red side of the generated SC with associated L-shaped histograms (see subplots 2), when a filter is used to isolate those fluctuations. We also stress that significant fluctuations can occur for normal pumping when the initial pulse spectrum lies close to the ZDW (see Fig. 5c) [55; 56].

Figure 5: Superposition of 200 simulations of the pulse spectrum after propagation in a fiber with a single ZDW pumped in anomalous dispersion (subplots a), a fiber with two ZDWs and pumped in the anomalous dispersion regime (subplots b), and a fiber pumped in the normal dispersion regime and where the broadening process is governed by fourth-order modulation instability (subplots c). Details of the fiber parameters, input pulses can be found in publications [START_REF] Kibler | Soliton and rogue wave statistics in supercontinuum generation in photonic crystal fibre with two zero dispersion wavelengths[END_REF] and [START_REF] Hammani | Soliton generation in a microstructured fiber by fourth order scalar modulation instability[END_REF] respectively. The results are obtained by the numerical integration of the generalized NLSE.

Optical rogue solitons

The main questions arising from the pioneering observations of rogue waves in optical systems are directed towards the physical nature of the extreme events and the mechanisms behind their emergence. A careful analysis of various configurations has now led to the conclusion that the most red-shifted events detected in the original experiments of Solli et al are fundamental solitons shifted in frequency by the intra-pulse Raman response of silica [START_REF] Mitschke | Discovery of the soliton self-frequency shift[END_REF]. As an example, Fig. 6 shows the spectrogram of an extreme event obtained in a SC driven by fourth-order modulation instability [START_REF] Hammani | Soliton generation in a microstructured fiber by fourth order scalar modulation instability[END_REF]. One can clearly identify the signature of the extreme event in the form of a transform-limited coherent soliton structure that emerges at long wavelengths. Because such giant solitons are fully coherent and persist once they form, they are now generally referred to as "rogue solitons" [START_REF] Dudley | Extreme events in optics: Challenges of the MANUREVA project[END_REF]. 5c). (b) Detail of the temporal structure of the most frequency red-shifted event (solid line) compared with the shape of a fundamental soliton. Details of the numerical simulations can be found in [START_REF] Hammani | Soliton generation in a microstructured fiber by fourth order scalar modulation instability[END_REF].

A physical process involved in the emergence of extreme solitons is the process of temporal collisions between the substructures generated by splitting of an initial pulse. When two ultrashort substructures collide, a temporal peak appears intermittently, thus reproducing one of the peculiar features of the infamous oceanic freak waves [START_REF] Kharif | Rogue waves in the ocean[END_REF]: their unexpected appearance and decay. Such collisions are favored by convective mechanisms such as the third-order dispersion or the Raman response of silica [START_REF] Taki | Thirdorder dispersion for generating optical rogue solitons[END_REF]. A dedicated care should be provided to their detection by a frequency offset filter that can in itself create an artifact [START_REF] Hage | Fiber-based device for the detection of low intensity fluctuations of ultrashort pulses[END_REF] and may not see these collision events directly [64; 65] as the emerging temporal spike of light does not correspond to the most red-shifted event. In the process of collision, an energy exchange also occurs, leading to the progressive growth of a giant soliton, where most of the energy of the system will eventually concentrate [66; 67]. It is worth noting here that fluctuations do not develop only on the long wavelength side of the SC, but also on the short wavelength edge of the supercontinuum with a statistical distribution that deviates from a Gaussian statistics [58; 68] as a result of pulse trapping by the redshifted solitons [START_REF] Nishizawa | Pulse trapping by ultrashort soliton pulses in optical fibers across zero-dispersion wavelength[END_REF] .

Spontaneous modulation instability and Akhmediev breathers

Whilst SC dynamics are now well-understood and rogue events can be explained through the prism of optical solitons [START_REF] Skryabin | Looking at a soliton through the prism of optical supercontinuum[END_REF], the temporal analysis of the fluctuations associated with anomalous pumping has led to reconsider the onset of SC generation where higher-order linear and nonlinear effects do not play an extensive role and pulse propagation can be described in the framework of the standard NLSE. In fact, the stage of MI that governs the initial spectral broadening can be described analytically within the framework of the Akhmediev breather formalism. Akhmediev breathers are particular solutions of the NLSE derived by Akhmediev and Korneev in 1986 which describe the single growth-return cycle of an initially weak sinusoidal modulation [START_REF] Akhmediev | Modulation instability and periodic-solutions of the nonlinear Schrödinger equation[END_REF]. Numerical simulations and experiments have indeed confirmed that the properties of SC at the early stages of propagation can be accurately described in terms of multiple breathers corresponding to the excitation of the entire MI gain bandwidth [START_REF] Dudley | Modulation instability, Akhmediev Breathers and continuous wave supercontinuum generation[END_REF]. In particular, the characteristics observed at the point of maximum temporal compression can be estimated by considering the properties of the breather corresponding to the frequency of the MI gain peak and whose spectrum exhibits a characteristic triangular shape on a log-log scale [START_REF] Dudley | Modulation instability, Akhmediev Breathers and continuous wave supercontinuum generation[END_REF].

Generation of highly coherent supercontinuum in the anomalous dispersion regime and observation of the Peregrine soliton

Seeded generation

The various examples of SC generation described in the previous section show that the control of the spectral dynamics, shape and stability and coherence properties is in general a complex issue.

In the normal dispersion regime, changing the temporal intensity or phase profile of the input pulse can provide a direct means of tailoring the continuum. On the contrary, in the anomalous dispersion regime, the SC dynamics is governed by solitonic effects that are more difficult to harness.

One way to limit the degradation of the SC coherence is to use very short segments of fiber or tapered fibers [5; 70]. An alternative solution is to reduce the spontaneous instabilities that may emerge in the first stages of propagation. In this paper, we do not consider the case when the pump itself is only partially coherent, which would require consideration on an incoherent modulational instability [START_REF] Hammani | Emergence of extreme events in fiber-based parametric processes driven by a partially incoherent wave[END_REF]. Here the pump is a coherent signal only affected by quantum noise and a small level of input amplitude fluctuations. By imposing an input seed located in the frequency band of the instabilities, one can expect that this deterministic seed will prevail over the spontaneous effects so that enhanced stability and coherence can be ensured.

Such an approach has been used in various configurations, in both numerical [71; 72] (Fig. 8(a)) and experimental [73; 74; 75] works in order to control the stability and spectral extent of the generated continuum (Fig. 8(b)). Other straightforward applications to optical parametric amplification and frequency conversion [START_REF] Solli | Seeded Supercontinuum Generation with Optical Parametric Down-Conversion[END_REF] as well as the all-optical generation of stable pulse trains with very high repetition rates [34; 77] have also benefited from the enhanced stability resulting from seeded parametric processes. An accurate spectral study has confirmed that the seeded generation can be well described by the mathematical formalism introduced by Akhmediev [START_REF] Hammani | Spectral dynamics of modulation instability described using Akhmediev breather theory[END_REF].

Observation of the Peregrine soliton in optical fiber

The control of MI dynamics shows that a judicious choice of excitation parameters and fibers can significantly improve the overall performance level of a broadband source by limiting the emergence of random rogue solitons. Significantly, the same kind of control can be used to generate the ultimate prototype of nonlinear rogue wave, the Peregrine soliton. This nonlinear structure has been first theoretically predicted in 1983 in hydrodynamics [START_REF] Peregrine | Water waves, nonlinear Schrödinger equations and their solutions[END_REF] but, up to 2010 and experiments in fiber optics, has never been demonstrated experimentally in any continuous physical media. The Peregrine soliton represents a limiting case of the Akhmediev breather for which the temporal localization is maximum [START_REF] Akhmediev | Waves that appear from nowhere and disappear without a trace[END_REF]. Consequently, this pulse over a continuous background has the particularity to appear from nowhere and to disappear without leaving a trace, thus constituting an excellent prototype of oceanic rogue waves. From an experimental point of view we have shown that a sinusoidal beating created by the temporal overlap of two continuous wave that are frequency shifted [START_REF] Kibler | The Peregrine soliton in nonlinear fibre optics[END_REF] or the direct intensity modulation of a continuous wave [START_REF] Hammani | Peregrine soliton generation and breakup in standard telecommunications fiber[END_REF] can reshape into a Peregrine soliton [START_REF] Erkintalo | Akhmediev breather evolution in optical fiber for realistic initial conditions[END_REF] . The obtained Peregrine soliton was accurately characterized using a frequency resolved optical gating device [START_REF] Kibler | The Peregrine soliton in nonlinear fibre optics[END_REF] or an optical sampling oscilloscope [START_REF] Hammani | Peregrine soliton generation and breakup in standard telecommunications fiber[END_REF]. Experimental results are well reproduced both numerically and analytically as can be seen in Fig. 9. The longitudinal study performed by a cut-back technique has confirmed the strong spatial localization (Fig. 9(b)), though some replicas are manifested for large propagation distances. Those replicas can be theoretically interpreted in terms of higherorder breather solutions through the Darboux transformations and linked to the process of higher order modulation instabilities [START_REF] Erkintalo | Higher-Order Modulation Instability in Nonlinear Fiber Optics[END_REF]. Note that the Peregrine is only the first order solution of a wider class of rational solitons that have been also numerically found in turbulent optical environments [85; 86].

Conclusions

We have provided an overview of several recent results on the stability of supercontinua and associated dynamics in normally and anomalously dispersive optical fibers. Normally dispersive fibers are very attractive for the generation of stable continua and different choices of input pulse characteristics enable a rich variety of reshaping processes within the fiber. In fibers with anomalous dispersion or fibers where spontaneous modulation instability processes are significant, the stability is much more difficult to control and the resulting SC is typically affected by large shot-to-shot fluctuations that favor the emergence of intense solitons on the red side of the spectrum and nonlinear pulse collisions. A fine analysis of the onset of the continuum generation has demonstrated that the Akhmediev breather formalism can provide new insight to understand and predict the early stages of propagation. Moreover, seeding the SC generation process with a suitably chosen continuous signal significantly improves the stability. The same concept can be applied to stimulate the generation of the Peregrine soliton, a limiting case of the Akhmediev breather, whose temporal and spatial localizations make it an excellent prototype of rogue waves that appear from nowhere and disappear without leaving a trace. More generally, the results presented in this paper are a further illustration of how optical fiber systems can provide useful experimental testbeds for the study of complex statistical hydrodynamics problems.

Figure 1 :

 1 Figure 1: Evolution of different initial pulses in a normally dispersive fiber: transform-limited pulse with hyperbolic secant, parabolic and triangular intensity profiles (subplots a, b, c respectively) and linearly chirped Gaussian pulse (subplot d). Subplots (1) show the longitudinal evolution of the spectral intensity profile, and subplots (2) show the temporal intensity at different stages of propagation. The initial pulses have a full-width at half maximum (FWHM) duration of 3 ps with an input peak power of 12 W and propagate in a highly non-linear fiber with a non-linear coefficient of 10 /W/km and a dispersion coefficient of -0.5 ps/nm/km. The results are obtained by numerical integration of the standard NLSE.

  of spectral recompression where, contrary to what might be expected based on simple considerations, SPM leads to energy concentration at the center of the pulse spectrum [29; 30; 31; 32] (Figs. 1(d) and 2(d)).

Figure 2 :

 2 Figure 2:Experimental results of the propagation of a hyperbolic secant pulse in a dispersion-shifted fiber, of a parabolic pulse in a highly nonlinear fiber followed by spectral slicing (the different channels are plotted), of a triangular pulse and of a Gaussian pulse with a parabolic phase (subplots a, b, c and d respectively). Details of the different experimental configurations can be found in[START_REF] Finot | Beneficial impact of wave-breaking on coherent continuum formation in normally dispersive nonlinear fibers[END_REF],[START_REF] Parmigiani | Ultra-flat SPM-broadened spectra in a highly nonlinear fiber using parabolic pulses formed in a fiber Bragg grating[END_REF],[START_REF] Verscheure | Pulse doubling and wavelength conversion through triangular nonlinear pulse reshaping[END_REF] and[START_REF] Fatome | All-fiber spectral compression of picosecond pulses at telecommunication wavelength enhanced by amplitude shaping[END_REF] respectively.

Figure 3 :

 3 Figure 3: Superposition of 200 simulations of the output temporal (a) and spectral (b) intensity profiles of a hyperbolic secant pulse (3 ps FWHM duration, 10 W peak power)propagating in a 500-m-long highly nonlinear fiber (nonlinear coefficient 10 /W/km, dispersion -0.5 ps/nm/km, dispersion slope 0.01 ps 2 /nm/km at 1550 nm). The results are obtained by numerical integration of the NLSE with third-order dispersion, including the quantum noise as well as 1% fluctuations on the input pump. The different shots are plotted in grey color and the average curve is plotted with a solid black line. Grey and black curves nearly coincide.

Figure 4 :

 4 Figure4: Superposition of 200 simulations of the ouput temporal (a) and spectral (b) intensity profiles of a secant hyperbolic pulse (FWHM duration of 3 ps, peak power of 10 W) propagating in a 500-m highly nonlinear fiber (nonlinear coefficient 10 /W/km, dispersion 0.5 ps/nm/km, dispersion slope 0.01 ps/nm/km at 1550 nm). Results are obtained by the numerical integration of the standard NLSE with third-order dispersion and include the quantum noise as well as 1% of the fluctuations on the input pump. The different shots are plotted in grey and the average curve is plotted with a solid black line.

Figure 6 :

 6 Figure6: (a) Spectro-temporal representation of a rogue event occurring in a SC driven by fourth-order modulation instability (cf. Fig.5c). (b) Detail of the temporal structure of the most frequency red-shifted event (solid line) compared with the shape of a fundamental soliton. Details of the numerical simulations can be found in[START_REF] Hammani | Soliton generation in a microstructured fiber by fourth order scalar modulation instability[END_REF].

Figure 7 :

 7 Figure 7: (a) Single shot NLSE simulations of the longitudinal evolution of a CW field undergoing spontaneous MI (subplot a1) and details of the temporal profile at 3.5 m propagation distance (subplot (a2)). The shaded region in the temporal trace is shown in detail in the rightmost figure comparing simulations (solid line) with the Akhmediev breather solution calculated for a modulation frequency corresponding to peak MI gain (dashed line). (b) Comparison between experiments (solid black line), numerical simulations using the full GNLSE (blue dashed line), numerical simulations using the NLSE only (red dashed line), and the calculated spectrum of the maximally-compressed Akhmediev breather (green open circles connected to the horizontal axis by straight lines from zero). Figures adapted from [10].

Figure 8 :

 8 Figure 8: (a1) Simulations results of SC generated by 20 ps pulses with modulation at 12.5 THz.Individual simulation results (gray traces) and the calculated mean from the ensemble (black line) are shown. The associated calculated coherence as defined in[START_REF] Dudley | Numerical simulations and coherence properties of supercontinuum generation in photonic crystal and tapered optical fibers[END_REF] is plotted on the right axis. (a2) Zoomed portion of the central part of the comb-like SC spectrum is displayed. Details of the numerical simulations are provided in ref[START_REF] Genty | Route to coherent supercontinuum generation in the long pulse regime[END_REF]. (b) Experimental set-up for the seeded generation of a continuum at telecommunication wavelengths. (c) Resulting experimental continuum recorded after 90m of highly nonlinear fiber. Details of the experimental parameters can be found in[START_REF] Barviau | Generation from continuous waves of frequency combs with large overall bandwidth and tunable central wavelength[END_REF].

Figure 9 :

 9 Figure 9: (a) Experimental set-up. CW : continuous wave, EDFA : erbium doped fiber amplifier, SMF : standard Single Mode Fiber ITU G652. Details of the experimental parameters are provided in [82]. (b) Longitudinal evolution of the temporal intensity profile as measured on the ultrafast sampling oscilloscope. Experiments (b1) are compared with numerical simulation of the NLSE (b2). (c) Temporal intensity profile at the point of maximum compression. The experimental results (red circles) are compared with the analytical form of the Peregrine soliton (solid black line). (d) Spectral intensity profile at the point of maximum compression. The experimental results (solid line) are compared to the results of numerical simulations (blue circles).

Acknowledgements

We would like to thank Antonio Picozzi, Miro Erkintalo, Frederic Dias, Nail N. Akhmediev for fruitful collaboration on rogue waves, Brandon G. Bale and Sergei Turitsyn for contributing to some aspects of pulse shaping in normally dispersive fibers, Francesca Parmigiani, Lionel Provost, Periklis Petroupoulos and Dave J. Richardson for their contribution to pulse shaping in normally dispersive fibers. This research was supported by the Agence Nationale de la Recherche (ANR MANUREVA, IMFINI, SUPERCODE, SOFICARS projects), by the Conseil Régional de Bourgogne (PARI Photcom and FABER of B. Kibler), by the FEDER, by the Ministère de l'Enseignement Supérieur et de la Recherche and by the Ministère des affaires étrangères (Alliance Scheme). The experimental work carried out at telecommunication wavelengths has benefited from the PICASSO Platform equipment.


Vincent Pw Scholtes

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Imo E Hoefer

Arjan H Schoneveld

Pieter R Stella

Pieter A F M Doevendans

Karlijn Jk Van Keulen

Dominique P V De Kleijn

Frans L Moll

Scholtes Vpw

De Vries Jppm

Hoefer Ie

Schoneveld Ah

Stella Pr

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De Kleijn Dpv

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Affiliations

Gerard Pasterkamp
email: g.pasterkamp@umcutrecht.nl

Karlijn Van Keulen

Imo Hoefer Pieter Stella

Toll Like Receptor 2 and 4 stimulation elicits an enhanced inflammatory response in human obese patients with atherosclerosis

Keywords: atherosclerosis, obesity, risk factors, innate immunity, Toll like Receptor, responsiveness

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Introduction

Atherosclerosis is considered as a generalized chronic inflammatory disorder of the vascular tree [START_REF] Ross | Atherosclerosis--an inflammatory disease[END_REF][START_REF] Stary | A definition of initial, fatty streak, and intermediate lesions of atherosclerosis. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis[END_REF]. Patients suffering from chronic inflammatory diseases, like systemic lupus erythematosus or rheumatoid arthritis have an increased risk for developing atherosclerosis [START_REF] Del Rincon | High incidence of cardiovascular events in a rheumatoid arthritis cohort not explained by traditional cardiac risk factors[END_REF][START_REF] Kao | Update on vascular disease in systemic lupus erythematosus[END_REF]. A potential clue for the underlying mechanisms of this increased prevalence, might be the chronic inflammatory state to which patients are subjected to. This enhanced systemic inflammatory state drives the successive activation of endothelial cells, leucocyte adherence and migration through the arterial wall leading to the production of pro-inflammatory cytokines. The relevance of smoking, diabetes, hypercholesterolemia and hypertension in the development of atherosclerosis is evident [START_REF] Wilson | Established risk factors and coronary artery disease: the Framingham Study[END_REF]. A possible explanation for the increased risk of atherosclerosis in the presence of these traditional risk factors, is the enhanced activation of the immune system.

Obesity is a worldwide increasing problem, especially in Western societies [START_REF] Who | Reducing risks, Promoting healthy life[END_REF][START_REF] Flegal | Prevalence and trends in obesity among US adults, 1999-2008[END_REF][START_REF] Lloyd-Jones | Heart disease and stroke statistics--2009 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee[END_REF]. Obesity is associated with an increased risk of acquiring traditional cardiovascular risk factors, potentiating the development of atherosclerosis and concomitant cardiovascular complications [START_REF] Flegal | Cause-specific excess deaths associated with underweight, overweight, and obesity[END_REF][START_REF] Kenchaiah | Obesity and the risk of heart failure[END_REF][START_REF] Yan | Midlife body mass index and hospitalization and mortality in older age[END_REF]. Obesity is also directly related to the development and progression of atherosclerosis [START_REF] Cassidy | Progression of subclinical coronary atherosclerosis: does obesity make a difference?[END_REF][START_REF] Fantuzzi | Adipose tissue and atherosclerosis: exploring the connection[END_REF][START_REF] Mcgill | Obesity accelerates the progression of coronary atherosclerosis in young men[END_REF][START_REF] Elkeles | The association of coronary calcium score and conventional cardiovascular risk factors in Type 2 diabetic subjects asymptomatic for coronary heart disease (The PREDICT Study)[END_REF]. Several articles provided evidence that adipose tissue is not just an energy storage depot but also a source of cytokine producing inflammatory cells [START_REF] Weisberg | Obesity is associated with macrophage accumulation in adipose tissue[END_REF][START_REF] Hotamisligil | Increased adipose tissue expression of tumor necrosis factor-alpha in human obesity and insulin resistance[END_REF][START_REF] Kern | The expression of tumor necrosis factor in human adipose tissue. Regulation by obesity, weight loss, and relationship to lipoprotein lipase[END_REF][START_REF] Berg | Adipose tissue, inflammation, and cardiovascular disease[END_REF][START_REF] Rocha | Obesity, inflammation, and atherosclerosis[END_REF].

Toll like receptors, which are part of the innate immune system, are pattern recognizing receptors forming the first line of defense, quickly reacting on evolutionary conserved pathogens [START_REF] Medzhitov | Toll-like receptors and innate immunity[END_REF]. But also endogenous ligands like extra domain A, heat shock proteins, fibronectin and free fatty acids are able to activate these receptors resulting into a pro-inflammatory cytokine response. TLR's are found in atherosclerotic tissue but also have shown to play a role in lipid uptake in macrophages and are expressed on circulating monocytes [START_REF] Edfeldt | Expression of toll-like receptors in human atherosclerotic lesions: a possible pathway for plaque activation[END_REF][START_REF] Kazemi | Adipocyte fatty acid-binding protein expression and lipid accumulation are increased during activation of murine macrophages by toll-like receptor agonists[END_REF]. We and others have shown the importance of these receptors in the initiation and progression of atherosclerosis [START_REF] Liu | Toll-like receptor 2 plays a critical role in the progression of atherosclerosis that is independent of dietary lipids[END_REF][START_REF] Schoneveld | Atherosclerotic lesion development and Toll like receptor 2 and 4 responsiveness[END_REF][START_REF] Vink | In vivo evidence for a role of toll-like receptor 4 in the development of intimal lesions[END_REF][START_REF] Ishikawa | Local expression of Toll-like receptor 4 at the site of ruptured plaques in patients with acute myocardial infarction[END_REF]. Responsiveness upon ligation of these receptors can be measured in whole blood, ex vivo. Previously, we showed that responsiveness altered with different severities of coronary artery stenosis [START_REF] Versteeg | Monocyte toll-like receptor 2 and 4 responses and expression following percutaneous coronary intervention: association with lesion stenosis and fractional flow reserve[END_REF]. Others showed that this response was elevated in patients having unstable angina pectoris or acute myocardial infarction, compared with patients with stable angina, [START_REF] Liuzzo | Enhanced response of blood monocytes to in vitro lipopolysaccharidechallenge in patients with recurrent unstable angina[END_REF][START_REF] Methe | Expansion of circulating Toll-like receptor 4-positive monocytes in patients with acute coronary syndrome[END_REF].

Considering the central role the Toll Like Receptor in atherosclerosis development, we hypothesized that cardiovascular risk factors are associated with an altered activation status of the innate immune system, reflected by differences in TLR responsiveness. We assessed the TLR response in patients with established atherosclerosis in two different territories: atherosclerosis of the coronary (percutaneous coronary intervention, PCI study) and of the carotid artery (carotid endarterectomy, CEA study) and related this to the presence of cardiovascular risk factors. In addition, we measured expression of the early activation marker CD11b on circulating monocytes.

We demonstrate that obesity is associated with an altered TLR response in patients with established atherosclerosis of the coronary or carotid artery.

Patients

Two cohorts of patients were analyzed for this study. Both studies were approved by the local medical committee board and informed consents were obtained. The first cohort consisted of 100 subsequent patients scheduled for Percutaneous Coronary Intervention (PCI study) at the University Medical Centre Utrecht. The second group consisted of 111 consecutive patients scheduled for carotid artery endarterectomy (CEA study) at the University Medical Centre Utrecht and at the St Antonius Hospital, Nieuwegein. This last patient group is part of the Athero-express Biobank study [START_REF] Verhoeven | Atheroexpress: differential atherosclerotic plaque expression of mRNA and protein in relation to cardiovascular events and patient characteristics. Rationale and design[END_REF].

Clinical data were obtained from patients medical file records and from pre-procedural filled-in questionnaires. Risk factors were scored as follows: Hypertension: the use of one or more antihypertensive drugs (> 3 months before intervention); Hypercholesterolemia: the use of statins for at least 3 months before intervention; Diabetes: the use of anti-diabetic medication (> 3 months before intervention); Positive family history of heart disease: > 1 family member with a cardiac event (MI, AP or CABG) < 60 years. Obese patients were classified as having a body mass index (BMI) > 25.0 and non-obese patients as having a BMI 25.0. Smoking status was derived from the preoperative intake questionnaire of the department of anesthesiology.

Blood sampling

Percutaneous Coronary Intervention (PCI) study. Whole blood samples were drawn in lithiumheparin (LH) and ethydelenediaminetetraacetic acid (EDTA) anticoagulated tubes before PCI. To prevent premature leucocyte activation all tubes were kept on ice until further processing. The effect of short term preservation on ice on cytokine production, was evaluated before initiation of this study. This short term preservation did not influence the experiments. 100 μl of blood was stimulated with 100 μl of the synthetic TLR2 ligand Pam3Cys-SK4 (P3C) (Novabiochem, Cambridge, MA, USA) at 500 ng/ml and 100 μl of TLR4 ligand LPS (derived of E. coli serotype O55:B5) (Sigma, St Louise, MO, USA) at 10 ng/ml. Samples were incubated overnight (o/n) at 37º C and 5% CO 2 . After o/n incubation, samples were centrifuged at 1000 rpm for 5 minutes and supernatants were collected and stored at -20ºC until further processing. As a surrogate of TLR2 and TLR4 response, TNF-alpha was measured in the supernatants using an enzyme linked immunosorbent assay (ELISA) according to the manufacturers protocol (Pelikine-compact, Sanquin, Amsterdam, the Netherlands). TNF-alpha levels were corrected for white blood cell count, which was measured using a hematology analyzer (Celldyn 1700, Abbott Diagnostics, IL, USA).

In EDTA anti-coagulated blood samples, TLR2, TLR4 and CD11b expressions on CD14 positive monocytes were determined by flow cytometry (Cytomics FC500, Beckman Coulter, CA, USA). Whole blood samples were stained for CD14 (PE-Cy5) combined with either TLR2 (FITC) and TLR4 (PE) or CD11b (FITC) (all Serotec, Oxford UK). TLR2, TLR4 and CD11b expression levels are referred to as mean fluorescence intensity (MFI) on CD14 positive monocytes. C reactive protein levels in serum were determined using a Nefelometer (Dade Behring, now Siemens Healthcare Diagnostics, Germany) , performed by the clinical chemistry laboratory of the UMC Utrecht hospital.

Carotid End-Arterectomy (CEA) study. Whole blood samples were drawn before incision, immediately after anesthesia. In a sub study the effect of anesthetics was investigated on TLR activation. The anesthetics did not influence the TNF-alpha levels (data not shown). Samples were stored in heparinized tubes and immediately processed at the laboratory. Blood samples were stimulated by using the same protocol as used in the PCI study, but instead of overnight incubation, samples were stimulated for 2 hours. As a surrogate of TLR2 and TLR4 response, TNF-alpha, IL6 and IL8 were measured in the supernatants using a multiplex immunoassay system according to the manufacturers protocol, (BioPlex 200, BioRad). TNF-alpha, IL6 and IL8 levels were corrected for white blood cell count. In heparin anti-coagulated blood samples, TLR2, TLR4, CD11b expression on CD14 positive monocytes were examined by flow cytometry (Cytomics FC500, Beckman Coulter, CA, USA). Whole blood samples were stained for CD14 (CD14ǦRPEǦCy5) combined with TLR2 (FITC) and TLR4 (PE) (Serotec, Oxford, UK ) and CD11b (CD11bǦPEǦCy7) (eBiosciences, San Diego, USA). TLR2, TLR4 and CD11b expression levels are referred to as MFI on CD14 positive monocytes. CD 11b expression on CD14 + cells was also measured after stimulation. 100 μl of heparinized whole blood was stimulated with 100 μl of: 1 PBS (negative control), 2 Pam3Cys (500ng/ml) (TLR2 stimulation) 3 LPS (100ng/ml) (TLR4 stimulation) and 4 Phorbol 12-myristate 13-acetate (PMA) (aspecific stimulation). Stimulated blood samples were incubated for 30 minutes at 37º C and 5% CO 2 .

Exclusion

In the PCI study, 22 patients were excluded for analysis: 9 patients suffering from acute myocardial infarction within 24 hours prior to PCI, 12 patients suffered from chronic inflammatory disorders receiving corticosteroid drugs and finally 1 patient was excluded because information about traditional risk factors was lacking. In the CEA study, 7 patients were excluded for analysis: 4 patients with chronic inflammatory disorder receiving corticosteroid drugs preoperatively and 3 patients, because white blood cell counts were not performed.

Statistics

TNF-alpha levels, mean fluorescence levels and CRP levels were not normally distributed. After ln transformation, the data was normally distributed. Differences between two independent variables were calculated with the independent sample T-test. Differences between 3 or more independent variables were calculated by using the one-way ANOVA test. Differences between 2 dichotomous values were calculated with the Chi-square test. Differences between 2 continuous variables were calculated by using the Spearman test. A p value < 0.05 was considered as statistically significant. Multivariate correction was performed by using a linear regression model. Gender, age, hypertension, hypercholesterolemia, diabetes, smoking status, positive family history for cardiovascular disease, hsCRP levels and BMI < 25 or BMI > 25 were analyzed with a linear regression model. In the CEA study, hsCRP levels were not available and positive family history for cardiovascular disease was left out the model because missing data of [START_REF] Kern | The expression of tumor necrosis factor in human adipose tissue. Regulation by obesity, weight loss, and relationship to lipoprotein lipase[END_REF] 22 patients in the PCI study and 7 patients in the CEA study were excluded from analysis (for details, see the methods section). 78 (PCI study) and 104 patients (CEA study) were used for analysis. Clinical characteristics are depicted at table 1 and S1.

In both studies, TNF-alpha was used as a specific marker for TLR response. Several authors have demonstrated the TLR specificity of this cytokine [START_REF] Hoshino | Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product[END_REF][START_REF] Takeuchi | Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components[END_REF]. Stimulation of whole blood samples with PBS did not result into detectable TNF-alpha levels and in addition stimulation with specific TLR2 and TLR4 agonists Pam3Cys and LPS resulted in a dose-dependent TNF-alpha release (data not shown).

In both studies, obese patients showed significantly higher TNF-alpha levels after stimulation of Toll Like Receptor 2, compared with non-obese patients: (median [IQR] pg/ml, adjusted p-value) [START_REF] Weisberg | Obesity is associated with macrophage accumulation in adipose tissue[END_REF].9 ). In the PCI study, we observed a trend towards lower levels of TNF-alpha following TLR2 and TLR4 stimulation for patients who were active smokers, compared with patients who do not smoke. However, this trend could not be observed in the CEA study (table 2). When taking other risk factors into account, we observed no differences in responsiveness between patients with or without these cardiovascular risk factors (table 2).

In the CEA study we also quantified the levels of IL6 and IL8 following TLR stimulation. Obese patients had compared with non-obese patients also increased levels of IL6 following TLR 2 and TLR 4 stimulation; (median [IQR] pg/ml, adjusted p-value) 7 [3 -30] As a marker for early activation, CD11b expression was measured at baseline in a consecutive subset of patients in both studies. In the PCI and CEA study, the flow cytometry measurements were included in the study protocol at a later time point. In both studies, obese patients revealed increased CD11b expression on CD14 positive monocytes, compared with non-obese patients, and these differences were significant in the PCI study (fig 2). In the CEA study we also measured CD11b expression after TLR 2 and 4 stimulation and again, obese patients showed increased expression of the activation marker CD11b (fig 2). After correction for risk factors the differences observed between obese and non-obese patients remained significant (p = 0.009 and p = 0.012 after stimulation with LPS and Pam3Cys resp). Differences between groups were not observed after stimulation with an aspecific stimulus, Phorbol 12-myristate 13-acetate (PMA) (fig 2). We did not observe differences in CD11b expression between patients with other established risk factors (table S2). In the PCI study we measured high sensitive C-reactive protein (hsCRP). Obese patients demonstrated higher levels of hsCRP (fig 3). Finally, TLR2 and 4 expressions on circulating monocytes were quantified in both studies. In both studies we did not observe differences in TLR 2 and 4 expression between obese and non-obese patients: median [IQR] PCI study: TLR2 expression 3.0 [2.4 -3.8] and 3.3 [2.8 -4.0] and TLR4 expression 2.4 [2.1 -3.6] and 2.8 [2.5 -3.5] for non-obese and obese patients respectively.CEA study: TLR2 expression 1.5 [0.9 -2.5] and 1.8 [1.0 -4.3] and TLR4 expression 0.3 [0.2 -0.5] and 0.5 [0.3 -0.7] for non-obese and obese patients respectively (table S3).

Discussion

Toll like receptor activation results in an innate immune response and plays an important role in the pathogenesis of atherosclerosis. An association between cardiovascular risk factors and the activation status of the innate immune system, which is reflected by an altered TLR responsiveness, can be suggested. We demonstrate in two cohorts of patients with established atherosclerosis, that obesity is a cardiovascular risk factor related with an increased TNF-alpha production by white blood cells following TLR activation.

Obesity and inflammation

While obesity was primarily seen as a lipid storage disease, it is nowadays recognized that obesity should be considered as a chronic inflammatory disorder [START_REF] Weisberg | Obesity is associated with macrophage accumulation in adipose tissue[END_REF][START_REF] Hotamisligil | Increased adipose tissue expression of tumor necrosis factor-alpha in human obesity and insulin resistance[END_REF][START_REF] Kern | The expression of tumor necrosis factor in human adipose tissue. Regulation by obesity, weight loss, and relationship to lipoprotein lipase[END_REF]. Several studies demonstrated that obese patients have increased levels of circulating biomarkers reflecting systemic inflammation [START_REF] Berg | Adipose tissue, inflammation, and cardiovascular disease[END_REF][START_REF] Visser | Elevated C-reactive protein levels in overweight and obese adults[END_REF]. High sensitive CRP is one of the most commonly studied biomarkers and is found to be elevated in obese patients [START_REF] Visser | Elevated C-reactive protein levels in overweight and obese adults[END_REF]. Our data also confirmed that obese patients, have increased levels of hsCRP (fig 3). Another activation marker of the immune cells is CD11b. CD11b forms together with CD18 the macrophage-1 antigen (Mac-1 or complement receptor 3) and is regarded as a key integrin in mononuclear cell adhesion towards activated endothelium [START_REF] Arnaout | Structure and function of the leukocyte adhesion molecules CD11/CD18[END_REF]. In both studies obesity was associated with increased CD11b expression on CD14 + monocytes (fig 3). In the CEA study, CD11b expression was also measured after TLR stimulation, which resulted in significant differences between obese and non-obese patients. Stimulation with the aspecific stimulus PMA (positive control) did not show any differences between obese and non-obese patients. Zhang et al. showed that physiological levels of Non Esterified free Fatty Acids (NEFA), which are elevated in obese patients, are able to up regulate CD11b expression on human monocytes and increase monocyte adherence on an adhesion assay [START_REF] Zhang | Elevated concentrations of nonesterified fatty acids increase monocyte expression of CD11b and adhesion to endothelial cells[END_REF]. CD11b is an important adhesion molecule, necessary for firm adhesion of the circulating monocyte to the activated endothelium. Enhanced expression of this integrin therefore might facilitate monocyte entry in atherosclerotic lesions. This could be one possible mechanism linking obesity with atherosclerosis.

Obesity and TLR response

In this study, obesity was the only risk factor that was associated with an increased TLR response. Obese patients have elevated levels of apolipoprotein CIII (apoCIII) and free fatty acids (FFA's) which are able to activate TLR 2 and 4 respectively [START_REF] Kawakami | Toll-like receptor 2 mediates apolipoprotein CIIIinduced monocyte activation[END_REF][START_REF] Shi | TLR4 links innate immunity and fatty acid-induced insulin resistance[END_REF]. TLR's are mainly expressed on leucocytes, but also adipocytes and macrophages in adipose tissue express these receptors. Release of apoCIII or FFA's, activate TLR 2 and 4 leading to a pro-inflammatory cytokine production. This might be an explanation of the increased levels of circulating biomarkers reflecting systemic inflammation in obese patients. Secondly, monocytes activated by these elevated levels of apoCIII or FFA's, will express more CD11b, and might adhere more easily to the activated endothelium. Because of their already activated state, upon stimulation, these monocytes will produce elevated levels of pro-inflammatory cytokines resulting in more severe atherosclerosis development.

Obese patients are also at increased risk for developing insulin resistance. In a recent review, Konner et al. summarized the abundant evidence that links TLR activation with insulin resistance [START_REF] Konner | Toll-like receptors: linking inflammation to metabolism[END_REF]. An important role for saturated FFA's causing insulin resistance in a TLR dependent manner was clearly highlighted. Saturated FFA's are able to activate inhibitor of nuclear factor (NF)-kB kinase (IKK) and c-Jun N -terminal kinase (JNK) in a TLR dependent manner. Activation of these important kinases not only results into transcription of pro-inflammatory cytokines (via NF-kB and AP-1) but also phosphorylates insulin receptor substrate (IRS) thereby causing insulin resistance. In a large genome wide association study, Weyrich et al. demonstrated an association between TLR4 D299G/T399l polymorphism and increased body fat and insulin resistance in 2 different cohorts, supporting the role of TLR4 as a molecular link between obesity and insulin resistance [START_REF] Weyrich | The D299G/T399I Toll-like receptor 4 variant associates with body and liver fat: results from the TULIP and METSIM Studies[END_REF].

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The enhanced responsiveness we observed in obese patients could not be explained by an elevated expression of TLR's on circulating monocytes. Therefore, other factors influencing TLR responsiveness might explain the increased responsiveness observed in obese patients. Schwarz et al. demonstrated that saturated free fatty acids, which are elevated in obese patients, are able to augment TLR induced cytokine production in a TLR independent manner [START_REF] Schwartz | Nutrient modification of the innate immune response: a novel mechanism by which saturated fatty acids greatly amplify monocyte inflammation[END_REF]. They showed that the saturated fatty acids lauric acid, palmitic acid or stearic acid are able to augment TLR2 and TLR4 response by stimulation of Protein Kinase C. Another possible explanation could be the effect of the antiinflammatory adipokine adiponectin, of which levels are decreased in obese patients [START_REF] Arita | Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity[END_REF]. Yamaguchi et al. demonstrated that adiponectin is able to inhibit TLR 2,4 and 9 induced NF-kB activation in murine macrophages [START_REF] Yamaguchi | Adiponectin inhibits Toll-like receptor family-induced signaling[END_REF]. In addition, another study showed decreased IL6 and TNF-alpha production of LPS stimulated porcine macrophages pretreated with adiponectin [START_REF] Wulster-Radcliffe | Adiponectin differentially regulates cytokines in porcine macrophages[END_REF]. Although the exact mechanism is still unknown, the decreased levels of adiponectin in obese patients might explain the observed increased responsiveness in obese patients.

Smoking and TLR response

In the PCI study, we observed a trend towards lower levels of TNF alpha following TLR stimulation in patients who are active smokers (table 2). When taking into account the time patients had stopped smoking there was a trend towards higher TNF alpha levels for patients who had stopped > 6 months or had never smoked (table 2). This trend could not be observed in the CEA study. Chen et al showed decreased TLR 2 and 4 responsiveness in alveolar macrophages isolated from active smokers compared with healthy controls, but failed to demonstrate decreased responsiveness of smokers PBMC's, following 6 hours of stimulation [START_REF] Chen | Tobacco smoking inhibits expression of proinflammatory cytokines and activation of IL-1R-associated kinase, p38, and NF-kappaB in alveolar macrophages stimulated with TLR2 and TLR4 agonists[END_REF]. We only observed the decreased TLR response in the PCI study, following a longer period of stimulation (overnight incubation). This might explain the differences we observed between our 2 studies and the study performed by Chen.

The strength of our study is that we demonstrated increased responsiveness in obese patients in two independent cohorts with established atherosclerosis of two different vascular territories. Another strength which is also a limitation of this study, is the different stimulation times used in both studies. It shows that differences can be observed between obese and non-obese patients despite changes in stimulation duration. On the other hand, we cannot state that we reproduced the results using an identical study design. Secondly, flowcytometry measurements were not performed after TLR stimulation in the PCI study. Finally, our study is descriptive and a control group of obese patients without cardiovascular disease is lacking. We can only speculate upon possible explanations for the increased responsiveness observed in obese patients. It would be of great interest to further explore this observation to see if healthy obese patients, without clearly established atherosclerosis, have also an increased TLR responsiveness. In addition, it would be interesting to see whether more specific markers of activation, like CD11c, are also elevated in obese patients.

Conclusion

Obese patients with established atherosclerosis show an increased whole blood TLR response and increased expression of the activation marker CD11b.

Clinical

  Science Immediate Publication. Published on 29 Mar 2011 as manuscript CS20100601 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100601 A c c e p t e d M a n u s c r i p t ϰ Material and Methods.

Clinical

  Science Immediate Publication. Published on 29 Mar 2011 as manuscript CS20100601 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100601 A c c e p t e d M a n u s c r i p t ϱ

  patients. All statistical analysis were performed with PASW statistics version 17 ( SPSS inc, Chigago, IL, USA) Clinical Science Immediate Publication. Published on 29 Mar 2011 as manuscript CS20100601 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100601 A c c e p t e d M a n u s c r i p t ϲ Results

  [7.7 -49.4] versus 7.5 [1.5 -19.2] and 14.6 [8.1 -28.4] versus 9.5 [6.1 -15.7] (p=0.008 and p = 0.015 for the PCI and CEA study, respectively) (fig 1). TNF-alpha levels in obese patients were also significantly higher compared with non-obese patients following TLR4 stimulation: 257.1 [129.0 -368.7] versus 180.9 [91.2 -236.6] (PCI study) p = 0.014 and not significantly enhanced in the CEA study: 209.4 [118.9 -340.2] versus 154.2 [105.4 -260.9] (p = 0.396). (fig 1

  vs 4 [2 -12] p = 0.045 and 123 [47 -240] vs 56 [31 -133] p = 0.054 respectively. IL8 levels were elevated in obese patients following TLR 2 stimulation, but not following TLR 4 stimulation. 68 [35 -147] vs 37 [23 -63] p = 0.042 and 560 [306 -857] vs 475 [226 -730] p = 0.729 respectively. (figure 4).

Clinical

  Science Immediate Publication. Published on 29 Mar 2011 as manuscript CS20100601 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100601 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited ϳ

Clinical

  Science Immediate Publication. Published on 29 Mar 2011 as manuscript CS20100601 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100601 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited

ClinicalFigure 1 2 3 4

 1234 Figure 1 Toll like receptor (TLR) response and obesity: TNF-alpha levels (ln transformed) are depicted on the y-axis with levels following TLR2 stimulation (with Pam3Cys) at the left and levels following TLR4 stimulation (with LPS) at the right. The grey circles represent obese patients, the white circles are non-obese patients. P-values are adjusted for cardiovascular risk factors. PCI study = percutaneous coronary intervention study, CEA study = carotid endarterectomy study

  52.5-69.7] 65.0[61.0-72.0]* 58.6[51.9-67.2]* 70.4[62.2-81.0] 73.7[66.3-79.9]* 69.3[61.7-74.6]* BMI (kg/m 2 ) median [IQR] 26.9[24.6-34.9] 24.0[22.6-24.7]** 28.7[26.6-32.4]** 26.2[24.1-29.7] 23.1[21.8-24.2]** 29.0[26.6-30.7]** A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited ϭϴ Table 2

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2011 The Authors Journal compilation © 2011 Portland Press Limited

This research was performed within the framework of CTMM, the Center for Translational Molecular Medicine (www.ctmm.nl), project CIRCULATING CELLS (grant 01C-102), and supported by the Netherlands Heart Foundation.

Supplementary table 1

Extra information on risk factors in both studies. Risk factors were scored using the following criteria: Hypertension: the use of one or more antihypertensive drugs (> 3 months before intervention); Hypercholesterolemia: the use of statins for at least 3 months before intervention; Diabetes: the use of anti-diabetic medication (> 3 months before intervention); Systolic and diastolic tension, glucose levels and cholesterol, HDL, TG and LDL levels of patients with or without hypertension, diabetes or hypercholesterolemia respectively, are depicted. There was no difference in systolic and diastolic tension between patients with or without antihypertensive medication. In both studies, patients on diabetic medication had compared with patients without diabetic medication, significantly higher blood glucose levels. Patients on statin therapy had compared with patients without statins significantly lower LDL levels. † from 4 patients systolic and diastolic pressure values could not be obtained. ‡ systolic and diastolic pressure values could not be retrieved from patients of the PCI study § glucose levels from 30 patients are missing || cholesterol, TG, HDL and LDL values from 53 patients are missing * p < 0.05 ** p < 0.010 *** p< 0.001 Supplementary table 2 Risk factors for atherosclerosis and CD11b expression on CD14 + monocytes at baseline. Median values and the interquartile range, together with number of patients, depicted between the brackets are provided for the different risk factors. PCI study = percutaneous coronary intervention study, CEA study = carotid endarterectomy study † from 18 patients no data could be obtained concerning family history of heart disease. These patients were excluded for this analysis. * p < 0.05 (*) p = 0.05 Supplementary table 3 Risk factors for atherosclerosis and TLR2 and TLR4 expression on CD14+ monocytes at baseline. Median values and the interquartile range, together with number of patients depicted between the brackets are provided for the different risk factors. PCI study = percutaneous coronary intervention study, CEA study = carotid endarterectomy study † from 18 patients no data could be obtained concerning family history of heart disease. These patients were excluded for this analysis.


Studies of infrasound production and perception by the Capercaillie (Tetrao urogallus):

A reply to Freeman and Hare. In their letter to the editor (Freeman and Hare, 2011), Freeman and Hare suggest that the technical procedures used in our study of infrasound responses in the Capercaillie (Tetrao urogallus) were improper and at least inadequate to answer the questions posed. We reject their comments unreservedly, for the following reasons:

1. Freeman and Hare (2011) assume that we utilized one particular kind of automotive loudspeaker for this study. In fact we used the "Infinity Kappa Sub" subwoofer designed for use in buildings (Harman Deutschland GmbH & Co. KG, Hünderstraße 1, D-74080 Heilbronn). This loudspeaker has far better technical specifications than Freeman and Hare (2011) assumed, i.e., "±3dB from 25 to 150Hz" and certainly does not show a spectrum that "rolls off dramatically at frequencies below 100Hz" (Freeman and Hare, 2011). Indeed, without appropriate levelling adjustments, this speaker has it strongest spectral component near 40Hz. The figure shows a spectrum of the "Flutter-jump" behaviour of a male Capercaillie played back using the Infinity Kappa Sub loudspeaker and as recorded with our equipment as described (Lieser et al., 2006). The spectral components recorded in the field in our first study (Lieser et al., 2005) are clearly present in the playback, the lowest harmonic component being near 9Hz. In the aviary used for playback studies, the achievable sound pressures are shown in figure 1.

2. Freeman and Hare (2011) inexplicably imply that we are unaware that "human hearing falls off below 40Hz" and therefore would have needed specialized equipment to detect the ultrasound. They thus completely ignore the details provided on the sound-recording equipment used in both studies. The microphone used had a flat spectrum from 3.15 Hz to 20 kHz and, under field conditions and with the amplifier and recording system used, was ample for sound pressures across this spectrum that exceeded 10 -20 dB SPL. As can be seen in the figure, the equipment clearly separated the harmonic spectral components 9Hz, 18Hz, 27Hz, etc. We do, however, agree fully with Freeman and Hare (2011) that "It is important to properly test the responses of Capercaillie to infrasound…" and respectfully suggest that this is in fact what we have already done.