N Elket

E Kendjo

M Thellier

L Assoumou

V Potard

A Taieb

I Tantaoui

E Caumes

R Piarroux

C Roussel

P Buffet

D Costagliola

Dr Stéphane Jauréguiberry
email: stephane.jaureguiberry@aphp.fr

French Artesunate

Working Group

Propensity score analysis of artesunate versus quinine for severe imported Plasmodium falciparum malaria in France

Keywords:

Background. Little is known on the use of artesunate compared to quinine for the treatment of imported malaria cases in non-endemic countries with high level of care. Therefore, we compared the two treatments in terms of mortality, hospital and intensive care unit discharge rates.

Methods. We analyzed the cohort of all severe imported malaria patients reported to the French National Reference Center from 2011 to 2017. After controlling for differences between quinine and artesunate treated individuals using the inverse probability of treatment weighting method, 28 day mortality rate was compared between the groups as well as hospital and ICU discharge rates using Kaplan Meier estimation and weighted Cox proportional hazard models.

Results. Overall, 1544 patients were enrolled. Fifty patients died, 18 in the quinine group (n=460) and 32 in the artesunate group (n=1084) corresponding to death rates of 3.9% 2.9% respectively. No difference was evidenced between quinine and artesunate neither in mortality nor in hospital discharge rate with HR= 1.03, CI 95% [0.47-2.25] and HR= 1.12, CI 95% [0.94-1.34] respectively. Artesunate was associated with a faster ICU discharge rate, HR = 1.18, CI 95% [1.02-1.36].

Conclusions.

In a country with a high level of care, artesunate was associated with a shorter length of stay in the ICU and this supports the actual therapeutic transition, however no difference was found neither in terms of mortality nor in hospital discharge rates between artesunate and quinine patients.

Key points

In a country with high level of health care, no difference was demonstrated neither in mortality nor in hospital dicharge rate between quinine and artesunate in the treatment of severe malaria. However patients treated with artesunate had a shorter length of stay in the ICU.

The WHO recommended intravenous artesunate (AS) as a first-line treatment for all forms of severe malaria worldwide since 2010 [START_REF] Who | The treatment of malaria[END_REF]. This followed two large randomized trials conducted in Asia and Africa that compared AS and intravenous quinine (Q) in the treatment of severe malaria showing an absolute reduction in death rates among adults and children in favor of artesunate [START_REF] Dondorp | (SEAQUAMAT) group. Artesunate versus quinine for treatment of severe falciparum malaria: a randomised trial[END_REF][START_REF] Dondorp | Artesunate versus quinine in the treatment of severe falciparum malaria in African children (AQUAMAT): an open-label, randomised trial[END_REF]. In May 2011, The French national drug Agency (ANSM) has granted a temporary authorization for use of AS as it was not available in France before and had no marketing authorization [START_REF]protocole d'utilisation thérapeutique et de recueil d'informations[END_REF]. In 2017, an update of the "2007 recommendations for the management of imported malaria", by the French society of Infectious Diseases (SPILF), endorsed and positioned artesunate as a first line treatment for severe malaria in France [START_REF]Prise en charge et prévention du paludisme d'importation[END_REF]. Studies performed in Western and endemic countries confirmed the efficacy of AS and showed an acceptable tolerance [START_REF]solution injectable -ATU nominative[END_REF][START_REF] Roussel | Artesunate to treat severe malaria in travellers: review of efficacy and safety and practical implications[END_REF]. Moreover, studies performed in Western countries highlighted the risk of an AS adverse event, which is post artesunate delayed hemolytic anemia (PADH) that appears during the 2nd or 3rd week after the end of treatment, showing the importance of follow-up [START_REF] Jauréguiberry | Postartesunate delayed hemolysis is a predictable event related to the lifesaving effect of artemisinins[END_REF].

Although AS tends to gradually replace quinine in the treatment of severe imported malaria in France, its use is still not mandatory and patients are still treated with Q, 70% in 2011 vs. 13% in 2015 according to the National reference French center of Malaria (NRC-M) 2017's annual report and there is no Good Manufacturing Practice-conform drug available yet. Moreover, little comparative data is available in high income countries like France comparing the use of AS instead of Q in managing severe imported malaria patients. Unfortunately, clinical randomized trials are no longer possible considering the previous published results confirming that AS saves more life than quinine and is better tolerated and considering the inadequate number of patients that can be included [START_REF] Dondorp | (SEAQUAMAT) group. Artesunate versus quinine for treatment of severe falciparum malaria: a randomised trial[END_REF][START_REF] Dondorp | Artesunate versus quinine in the treatment of severe falciparum malaria in African children (AQUAMAT): an open-label, randomised trial[END_REF][START_REF] Sinclair | Artesunate versus quinine for treating severe malaria[END_REF]. However, the effect of AS on reducing the death's rates was rigorously demonstrated only in endemic countries where there is no existing or poorly available ICU facilities. Questions remain on the generalization of those findings especially that the supportive care and ICU facilities as well as patient's characteristics may differ significantly in high income countries from endemic ones. A recently published study carried out between 2006 and 2010 on 155 severe malaria cases, treated with Q in intensive care unit (ICU) in France showed a mortality rate of 5% [START_REF] Bruneel | Imported falciparum malaria in adults: host-and parasite-related factors associated with severity. The French prospective multicenter PALUREA cohort study[END_REF] whereas the surveillance of AS in France, showed a mortality rate of 3% [START_REF]solution injectable -ATU nominative[END_REF]. However, these results are not comparable and the real impact of AS compared to quinine remains unknown.

Our study compared AS with Q in the treatment of severe imported malaria between 2011 and 2017 to determine the impact of using AS in terms of mortality, hospital and ICU discharge rates in high level of health-care.

METHODS

Study population

We analyzed a historical cohort of all Plasmodium falciparum severe imported malaria cases reported from 2011 to 2017 to the NRC-M, a network of 110 hospitals throughout France. All data were prospectively recorded. We used the case definition of the national French guidelines of the SPILF for management of imported malaria cases in 2007 [START_REF] Spilf | Recommendations for clinical practice. Management and prevention of imported Plasmodium falciparum malaria. (Revision 2007 of the 1999 Consensus conference)[END_REF]. A severe case of malaria was defined by a positive blood smear for the asexual form of P. falciparum and the presence of one or more severity criteria at admission (Supplementary Text S1). All patients that received AS or Q as first line treatment at the time of diagnosis of severe malaria were eligible.

Statistical analysis

The primary objective was to compare 28 day mortality rates between individuals receiving AS and Q. The secondary and third objectives were the hospital and ICU discharge rates following treatment administration. Analyses were on an intention-to-treat (ITT) basis, patients were analyzed depending on which arm they were assigned to initially.

Missing data

All variables from the cohort were prospectively recorded, however some data were missing. Multiple imputation using Chained Equations approach (MICE) was used to fill in missing data [START_REF] White | Multiple imputation using chained equations: Issues and guidance for practice[END_REF]. Ten imputations (M=10) were chosen to obtain valid inference and reduce sampling variability resulting from the imputation process. Variables with a missing rate above 15% were excluded and the outcomes were included in the imputation model. All 10 datasets were analyzed and combined using Rubin rules [START_REF] Rubin | Multiple imputation for nonresponse in surveys[END_REF].

Propensity score

Differences between AS and Q groups were analyzed using univariable logistic regressions. To avoid a statistically significant difference in outcome between the two groups of patients that may be observed in the absence of treatment effect but due to a difference in initial prognosis between the two groups compared we use a propensity score analysis approach (PS) [START_REF] Rosenbaum | The central role of the propensity score in observational studies for causal effects[END_REF]. The PS for each subject was defined as the conditional probability of receiving AS given the patient's individual baseline characteristics at admission. The index date for all the analyses was the day the patients start the treatment (day 0). PS was estimated from a logistic regression model that included factors predictive to the outcomes whether they were associated with the exposure or not as well as factors that might have influenced the treatment choice [START_REF] Austin | Moving towards best practice when using inverse probability of treatment weighting (IPTW) using the propensity score to estimate causal treatment effects in observational studies[END_REF]. Factors associated with the outcomes were tested using univariable hazards models. All potential confounders that are known in the literature were also included [START_REF] Hcsp | Place de l'artésunate injectable dans le traitement du paludisme grave de l'adulte et de l'enfant en France[END_REF][START_REF] Bruneel | Severe Imported Falciparum Malaria: A Cohort Study in 400 Critically Ill Adults[END_REF][START_REF] Legros | Risk factors for imported fatal Plasmodium falciparum malaria, France, 1996-2003[END_REF][START_REF] Kurth | Intravenous Artesunate Reduces Parasite Clearance Time, Duration of Intensive Care, and Hospital Treatment in Patients With Severe Malaria in Europe: The TropNet Severe Malaria Study[END_REF]. A different PS was calculated for each outcome as the number of selected subjects changed each time.

Variables included in the PS model are described in Supplementary Text S0.

Propensity scores were then used by inverse probability of treatment weighting method (IPTW) [START_REF] Austin | Moving towards best practice when using inverse probability of treatment weighting (IPTW) using the propensity score to estimate causal treatment effects in observational studies[END_REF][START_REF] Crowson | The Basics of Propensity Scoring and Marginal Structural Models[END_REF]. The stabilized weights were calculated as p/PS for individuals who received AS, and as (1 -p)/(1 -PS) for individuals who received Q, where p is the overall marginal prevalence of treatment exposure [START_REF] Crowson | The Basics of Propensity Scoring and Marginal Structural Models[END_REF]. The balance in characteristics between the 2 groups in the original and weighted samples was checked using standardized differences. Absolute standardized differences of less than 10% are sufficient to demonstrate achieved balance [START_REF] Austin | Moving towards best practice when using inverse probability of treatment weighting (IPTW) using the propensity score to estimate causal treatment effects in observational studies[END_REF][START_REF] Crowson | The Basics of Propensity Scoring and Marginal Structural Models[END_REF]. Propensity score analysis was performed in each imputed dataset. The treatment effect estimates from each imputed dataset were then combined to obtain an overall estimate [START_REF] Leyrat | Propensity score analysis with partially observed covariates: How should multiple imputation be used?[END_REF].

Endpoints

In order to compare the mortality in the two groups, weighted Kaplan Meier estimates were calculated for each group, 28 days after the index date. Weighted Cox proportional hazards models were estimated, where the only variable included in the model was the treatment and patients in the Q group served as reference.

As for the second and the third objectives, a propensity score was calculated for subjects with observed data on length of stay in hospital and in ICU after performing analyses to make sure patients with missing and observed length of stay were comparable. In these analyses, the maximum observed length of stay in hospital and in ICU was assigned to patients who died corresponding to 66 days and 70 days respectively. Weighted Kaplan Meier estimates as well as Cox proportional hazards models were used to compare hospital and ICU discharge rates in the 2 treatment groups.

Ethical considerations

Ethical considerations are described in Supplementary Text S0.

RESULTS

The cohort comprised 1544 patients with severe Plasmodium falciparum malaria between 2011 and 2017 (Figure 1). The majority of cases contracted the disease in west and central Africa (92.5%). The most common severity criterion was hyperparasitemia (59%), followed by cerebral malaria (38%) and the median number of severity criteria was 2 [START_REF] Dondorp | (SEAQUAMAT) group. Artesunate versus quinine for treatment of severe falciparum malaria: a randomised trial[END_REF][START_REF] Dondorp | Artesunate versus quinine in the treatment of severe falciparum malaria in African children (AQUAMAT): an open-label, randomised trial[END_REF]. The total number of patients having only a hyperparasitemia (>4%) as a severity criterion was 257/1544 (16.6%): 86 in quinine group (18.7%) and 171 in artesunate group (15.8%). The characteristics of the 2 groups are described in Table 1.

Missing data

In total 7 variables were imputed: the missing data percentage was 2.4% for region of malaria acquisition, 7.9% for parasitemia, 9.6% for platelet count, 10.7% for WBCs count, 9.5% for chemoprophylaxis, 10.1% for immunosuppression, 10.8% for patient's origin. The following variables had a high missing data rate, thus, they weren't included in the imputation model: length of travel 42%, presence of comorbidities >70% except immunosupression, reason of travel 25%.

Risk factor for death

A higher risk of death was observed in non-Africans patients, patients older than 60 years old and hyperparasitemic patients with a parasitemia level above 10% in univariable hazard models. Results also showed that the following severity criteria (cerebral malaria, circulatory collapse, respiratory distress, abnormal bleeding, acidosis, hyperlactatemia >5 mmol/L, hypoglycemia <2.2 mmol/L, creatininemia >265 micromol/L and clinical jaundice) were associated with death. Being treated in a center with an annual activity above 12 malaria cases per year was a protective factor (Supplementary Table S1).

Propensity score Propensity score's distribution before using the IPTW method for each outcome is displayed in Supplementary Figure S1. Absolute standardized differences after weighting were below 10% for all characteristics showing that reasonable balance has been achieved (Supplementary Figure S2).

Mortality rate

Among the 1544 reported cases, 50 died: 32 patients (2.9%) in the AS group and 18 (3.9%) in the Q group. Two more patients in the Q group died at day 36 and day 40, both were censored at day 28 in the survival analysis. Among all deaths, 82% occured while receiving first line treatment while 18% have switched treatments. In the Q group, 140 patients had switched to artesunate, 6 of all the patients who died in the Q group were among those switchers wheareas 8 patients in the AS group had switched to Q with only one death occuring after switching.

The estimated weighted hazard ratio after multiple imputation and IPTW was HR= 1.03, 95%CI [0.47-2.25], p= 0.923, with no evidence for difference between the two groups with regard to the mortality rate. Figure 2A displays estimated weighted Kaplan Meier estimates.

Hospital discharge rate

In total 960 patients were analyzed, 255 in the Q group and 705 in the AS group (Supplementary Table S2). Older age, non-African origin, immunosuppressed patients, platelet count <10 G/L and severity criteria as neurological failure, respiratory distress, abnormal bleeding, acidosis, hyperlactatemia >5mmol/L, jaundice, renal failure and a hyperparasitemia level >10% were factors associated with a slower discharge rate when tested in univariable analyses. Admission in year 2016-2017 and male sex were associated with a faster discharge rate. The following variables, endemic region, presence of repeated convulsion and hemoglobin level < 7g/L were not associated with the outcome in univariable analysis, thus were not included in the PS model. Weighted proportionnal hazard ratio was 1.12, 95%CI [0.94 -1.34], p=0.212, with no statistical evidence of difference in hospital discharge rates between the 2 treatment groups (Figure 2B). Median length of hospital stay was 6 [START_REF]protocole d'utilisation thérapeutique et de recueil d'informations[END_REF][START_REF]Prise en charge et prévention du paludisme d'importation[END_REF][START_REF]solution injectable -ATU nominative[END_REF][START_REF] Roussel | Artesunate to treat severe malaria in travellers: review of efficacy and safety and practical implications[END_REF][START_REF] Jauréguiberry | Postartesunate delayed hemolysis is a predictable event related to the lifesaving effect of artemisinins[END_REF][START_REF] Sinclair | Artesunate versus quinine for treating severe malaria[END_REF][START_REF] Bruneel | Imported falciparum malaria in adults: host-and parasite-related factors associated with severity. The French prospective multicenter PALUREA cohort study[END_REF] days in Q group and 5 [START_REF]protocole d'utilisation thérapeutique et de recueil d'informations[END_REF][START_REF]Prise en charge et prévention du paludisme d'importation[END_REF][START_REF]solution injectable -ATU nominative[END_REF][START_REF] Roussel | Artesunate to treat severe malaria in travellers: review of efficacy and safety and practical implications[END_REF] days in the AS group.

ICU discharge rate

This analysis was performed on 916 patients with 765 patients admitted in the ICU (Supplementary Table S3). The same factors associated with a slower hospital discharge rate were associated with a slower ICU discharge rate in univariable analyses except abnormal bleeding and immunosuppression. Year at admission was a variable that highly differentiated the groups and was weakly associated with the ICU discharge rate (p=0.048) so we excluded it from the propensity score model and we included it with the treatment variable in the final weighted Cox model. Patients on AS were discharged faster from the ICU compared to Q. A median of 2 [START_REF] Who | The treatment of malaria[END_REF][START_REF] Dondorp | (SEAQUAMAT) group. Artesunate versus quinine for treatment of severe falciparum malaria: a randomised trial[END_REF][START_REF] Dondorp | Artesunate versus quinine in the treatment of severe falciparum malaria in African children (AQUAMAT): an open-label, randomised trial[END_REF] days was observed in AS group and of 3 [START_REF] Dondorp | (SEAQUAMAT) group. Artesunate versus quinine for treatment of severe falciparum malaria: a randomised trial[END_REF][START_REF] Dondorp | Artesunate versus quinine in the treatment of severe falciparum malaria in African children (AQUAMAT): an open-label, randomised trial[END_REF][START_REF]protocole d'utilisation thérapeutique et de recueil d'informations[END_REF][START_REF]Prise en charge et prévention du paludisme d'importation[END_REF] days in Q group. Weighted proportionnal hazard ratio was 1.18, 95%CI [1.02 -1.36], p=0.03 (Figure 2C).

DISCUSSION

To the best of our knowledge, this observational study is the largest to compare Q and AS on mortality, hospital and ICU discharge rates in malaria non-endemic countries with highlevel of care. No difference was evidenced neither in mortality nor in total length of stay in the hospital between the 2 treatment groups, while artesunate was associated with a shorter length of stay in the ICU compared to quinine.

Very little comparative data on the use of either treatment was available for severe malaria cases in non-endemic countries. Four non-randomized studies provided comparative information in the context of severe imported malaria [START_REF] Kurth | Intravenous Artesunate Reduces Parasite Clearance Time, Duration of Intensive Care, and Hospital Treatment in Patients With Severe Malaria in Europe: The TropNet Severe Malaria Study[END_REF][START_REF] Mccarthy | Severe malaria in Canada, 2001-2013[END_REF][START_REF] Rolling | Artesunate versus quinine in the treatment of severe imported malaria: comparative analysis of adverse events focussing on delayed haemolysis[END_REF][START_REF] Eder | Intravenous artesunate versus intravenous quinine in the treatment of severe falciparum malaria: a retrospective evaluation from a UK centre[END_REF]. They all showed benefits of using artesunate over quinine. Kurth, et al. and Eder, et al. also showed a reduction in the time of parasitic clearance in favor of artesunate (75h vs. 96h) and (65h vs. 85 h) respectively. Rolling et al. showed a better tolerance with only postdelayed hemolytic anemia (PADH) reported as an adverse event for artesunate patients, while 71% of patients under quinine experienced hypoglycemia, cardiotoxicity or hearing disturbance. None of the previous studies in non endemic countries reported any difference in terms of mortality. The number of patients included was limited and statistical methods employed were simple.

Mortality rates reported in our study were very low in both the AS and the Q group (2.9% vs. 3.9% respectively) reflecting the importance of high level of care in the management of severe malaria cases. Estimated hazard ratio close to 1 and narrow confidence interval shows that a very large number of individuals would be needed to show a significant difference in mortality between the treatment groups. A study including 155 events which is 3 times more deceased patients might be needed to show a 1% difference in mortality between the groups with a power of 80% and clinical trials are no longer justified considering the previous published results and with regard to the number of patients that can be included in non-endemic countries. Moreover differences in mortality previously published between Q and AS patients were observed in endemic countries [START_REF] Dondorp | (SEAQUAMAT) group. Artesunate versus quinine for treatment of severe falciparum malaria: a randomised trial[END_REF][START_REF] Dondorp | Artesunate versus quinine in the treatment of severe falciparum malaria in African children (AQUAMAT): an open-label, randomised trial[END_REF], it is expected that the difference in mortality rates would be smaller in developed countries with high-level of care. Although AS did not reduce the total length of stay in the hospital, patients on AS were discharged faster from the ICU compared to Q. Shorter length of stay in the ICU is still beneficial in reducing the resources spent for patient's care per day. In France, the cost of a day spent in the ICU is of 2816 euros (www.aphp.fr). Besides, although the average cost of AS is currently higher than Q, the latest is administered 3 times daily while AS is administered once daily which is more convenient for patients as well as healthcare professionals taking care of them. This is supported by an economic evaluation of AS in south Asia that showed that the use of AS was cost-effective [START_REF] Lubell | Cost-effectiveness of artesunate for the treatment of severe malaria[END_REF].

One of the main strengths of our study is the large sample size compared to others as participants were enrolled from 110 centers participating to the national surveillance network for malaria (NRC-M) throughout France, which increases the robustness of our findings. The use of IPTW method assured the comparability of the groups on measured confounders such as older age, non-African origin, presence of neurological impairment, respiratory distress, circulatory collapse, abnormal bleeding, acidosis, hyperlactatemia >5 mmol/L, hypoglycemia <2.2 mmol/L, renal failure, platelet count below 10 G/L and hyperparasitemia above 10%. All those factors were associated to death in univariable analysis which is consistent with literature [START_REF] Bruneel | Severe Imported Falciparum Malaria: A Cohort Study in 400 Critically Ill Adults[END_REF][START_REF] Legros | Risk factors for imported fatal Plasmodium falciparum malaria, France, 1996-2003[END_REF]. Adding the variable annual activity of the center in the propensity score allowed us to take in consideration the indication bias since centers with a high number of cases admitted per year switched faster to artesunate.

Being treated in a center receiving more than 12 malaria cases per year was a protective factor from death in univariable analysis. We assume that such centers do better, mainly due to a better experience in handling patients with severe malaria. In our work African patients had a higher probability to receive AS. This could be explained by the fact that the African immigrant's population usually tend to live near the biggest cities in France and AS was introduced faster in large hospitals than in smaller ones. The time factor was also taken into consideration as most of patients who received Q were in early stages of the study while the number of patients that received AS increased gradually by time specially after 2014. Nevertheless this study had limitations. Imported malaria is not a mandatory notifiable infectious disease in France and cases out the NRC-M network might have been missed. Moreover the surveillance system relies on motivation of physicians and parasitologists to report data to the NRC-M, consequently available data were not complete. In addition, propensity score allows to control the two groups only on measured confounders which is a limitation of this approach. However, most important confounding factors were taking into account in our study except for the presence of other comorbidities than being immunocompromised, this was due to lack of data on this factor in our database. As this study was based on the intention-to-treat principle, patients who had switched treatment were analyzed in their initial group and this might have minimized the difference observed between the groups on measured endpoints, however data on time at switch was very limited. Another limitation of this study was that it didn't compare safety with regard to the treatment received. However, AS use was proved to be safe and well tolerable in France and other non-endemic countries with less frequent episodes of hypoglycemia and cardiotoxicity despite reported cases of PADH [START_REF] Roussel | Artesunate to treat severe malaria in travellers: review of efficacy and safety and practical implications[END_REF][START_REF] Jauréguiberry | Postartesunate delayed hemolysis is a predictable event related to the lifesaving effect of artemisinins[END_REF][START_REF] Jaureguiberry | Delayed-onset hemolytic anemia in patients with travel-associated severe malaria treated with artesunate, france, 2011-2013[END_REF].

In conclusion, this work suggests that a high level of intensive care can achieve a very low mortality rate regardless of the curative treatment. In view of the good tolerance of artesunate and its ease of use with regard to quinine, the current therapeutic transition is strengthened. Moreover, artesunate reduces the length of stay in ICU and this in turns supports the actual therapeutic transition happening in France and advocates for the implementation of ICU in developing countries. As a second line treatment in artesunate group: 151 patients have received non-artemisinin based combination therapy such as Atovaquone + Proguanil (Malarone®), Chloroquine (Nivaquine®), Halofantrine (Halfan®), Doxycycline (Doxypalu®), Méfloquine (Lariam®) and Quinine per os, 573 have received artemisinin-based combination therapy such as Artemether + Lumefantrine (Coartem®, Riamet®), Dihydroartémisinine + pipéraquine (Eurartésim®) and Artemether IM (Paluther®) and 8 patients have switched to quinine while 320 haven't received a second line treatment or data was missing for them. In the quinine group, as a second line treatment, 107 have received non-artemisinin based combination therapy, 44 have received artemisinin based combination therapy and 140 patients have switched to artesunate while 169 haven't received a relay. Two patients died after day 28 thus were censored in survival analysis. Misclassified patients are those who received the 2 treatments, but had missing data on criteria and dosing at admission.

Notes

Figure 1 .

 1 Figure 1. Flowchart of treated patients, French cohort 2011-2017.

Figure 2 .

 2 Figure 2. Kaplan Meier plots shows the time since initiation of the treatment to A.death, B. discharge from the hospital, C.discharge from the ICU in quinine and artesunate group with quinine group used as reference

Acknowledgments

*Members of the French Artesunate Working Group (FrAWG) and NRC-M are listed in supplementary material Text S2. Laboratoire d'Excellence GR-Ex, Université des Antilles et de la Réunion.

Financial support None

Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.


Aude Chiot

Christian S Lobsiger

Séverine Boillée
email: severine.boillee@upmc.fr

New insights on the disease contribution of neuroinflammation in ALS

Keywords: Amyotrophic Lateral Sclerosis (ALS), neuroinflammation, lymphocytes, monocytes, mast cells

Purpose of review: Amyotrophic Lateral Sclerosis (ALS) is a degenerative motor neuron (MN) disease with a strong neuroinflammatory component. This review summarizes how the connection between neurodegeneration and the immune system is strengthened by new discoveries from ALS genetics and the analysis of subpopulations of immune cells in ALS.

Recent findings:

Recent genes identified in ALS encode for proteins with direct immune roles, that when mutated lead to deregulation of immune functions, potentially influencing the disease. Although, neuroinflammation in the CNS of ALS has been well documented, new evidence suggests also direct malfunctions of immune cells in the CNS and at the periphery: While CD4+ T regulatory lymphocytes are protective in ALS, their number and function are altered over the disease course. CD8+ T cells are detrimental for MNs in the CNS, but show some protective roles at the periphery. Likewise, presence of mast cells in muscles of ALS models and patients, and impairments of monocyte functions reveal potential new players in ALS disease progression.

Summary:

Although MN degeneration is considered the prime event in ALS, dysfunctions in immune processes can impact the disease, highlighting that targeting specific immune components is a strategy for developing biomarkers and ultimately new drugs.

Introduction

Amyotrophic Lateral Sclerosis (ALS) is the most common adult onset motor neuron (MN) disease, characterized by the degeneration of upper and lower MNs. ALS is a heterogeneous disease, multifactorial, with some known genetic causes, but for the majority of cases, multigenetic and probable environmental causes are likely present [START_REF] Phukan | The syndrome of cognitive impairment in amyotrophic lateral sclerosis: A population-based study[END_REF]. To date, mutations in more than 25 genes, mainly autosomal dominant, have been linked to ALS, the major genes being C9orf72, SOD1, TARDBP and FUS/TLS (http://alsod.iop.kcl.ac.uk) [START_REF] Millecamps | TARDBP, and FUS mutations in familial amyotrophic lateral sclerosis: genotype-phenotype correlations[END_REF][START_REF] Brown | Amyotrophic Lateral Sclerosis[END_REF]. A hallmark of both familial and sporadic ALS, is the presence of reactive immune cells in postmortem tissues, with microglial cells, the macrophages of the CNS, found in the vicinity of degenerating MN cell bodies and axonal tracts [START_REF] Engelhardt | IgG reactivity in the spinal cord and motor cortex in amyotrophic lateral sclerosis[END_REF]. Microglial activation (as part of the neuroinflammatory response) has also been correlated to MN damage in ALS patients [START_REF] Turner | Evidence of widespread cerebral microglial activation in amyotrophic lateral sclerosis: An [11C](R)-PK11195 positron emission tomography study[END_REF][START_REF] Brettschneider | Microglial activation correlates with disease progression and upper motor neuron clinical symptoms in amyotrophic lateral sclerosis[END_REF].

To understand the role of these immune cells during the disease course, ALS mouse models have been extensively used, mostly mice expressing mutant Cu/Zn Super oxide Dismutase (SOD1), as they remained for the longest time the only model with clear recapitulation of progressive MN degeneration, and only in most recent time, alternative mouse models start to appear, although with variable efficiency and usability. Importantly, these studies with mutant SOD1 mice strongly suggested that ALS is a non-cell autonomous disease, where certain, but not all (non-neuronal) cell-types surrounding MNs contribute to disease toxicity, in particular and the focus of this review, microglial cells [START_REF] Boillée | Onset and progression in inherited ALS determined by motor neurons and microglia[END_REF][START_REF] Wang | The effect of mutant SOD1 dismutase activity on non-cell autonomous degeneration in familial amyotrophic lateral sclerosis[END_REF][START_REF] Beers | Wild-type microglia extend survival in PU . 1 knockout mice with familial amyotrophic lateral[END_REF]. Interestingly, while downregulating mutant SOD1 in MNs delayed disease onset, in microglial cells, it influenced disease progression [START_REF] Boillée | Onset and progression in inherited ALS determined by motor neurons and microglia[END_REF][START_REF] Wang | The effect of mutant SOD1 dismutase activity on non-cell autonomous degeneration in familial amyotrophic lateral sclerosis[END_REF][START_REF] Beers | Wild-type microglia extend survival in PU . 1 knockout mice with familial amyotrophic lateral[END_REF]. This specificity is of prime importance and led to three insights: (i) consistent with mutant SOD1 expressing microglial cells not being able, on their own, to kill control MNs in mice [START_REF] Beers | Wild-type microglia extend survival in PU . 1 knockout mice with familial amyotrophic lateral[END_REF], ALS remains primarily a MN disease, and microglial cells act by reacting to neurodegeneration; (ii) but it also indicates that reactive microglial cells can contribute to MN degeneration; and thus, (iii) it suggests that targeting microglial cells to slow disease progression could be effective, even in the sporadic ALS population. Microglial cells are part of the myeloid lineage, but they have a different developmental origin than most other tissue macrophages and blood monocytes. Other immune cells coming from the lymphoid lineage take also part in the disease. While depletion of B cells did not impact ALS disease in mice, specific deletion of CD4+ or CD8+ T lymphocytes has emphasized their opposite impact, detailed thereafter [10-14**].

When ALS genes show immune functions

C9orf72 is the major ALS-linked gene in the western world, characterized by a hexanucleotide expansion in its intronic region. Following this discovery, several mouse lines deleted for the C9orf72 ortholog were produced (C9orf72 KO mice), to understand the unknown function of C9orf72 and assess a possible loss-of-function mechanism [START_REF] Atanasio | C9orf72 ablation causes immune dysregulation characterized by leukocyte expansion, autoantibody production, and glomerulonephropathy in mice[END_REF][START_REF] Burberry | Loss-of-function mutations in the C9ORF72 mouse ortholog cause fatal autoimmune disease[END_REF][START_REF] Jiang | Gain of Toxicity from ALS/FTD-Linked Repeat Expansions in C9ORF72 Is Alleviated by Antisense Oligonucleotides Targeting GGGGCC-Containing RNAs[END_REF][START_REF] O'rourke | C9orf72 is required for proper macrophage and microglial function in mice[END_REF][START_REF] Sudria-Lopez | Full ablation of C9orf72 in mice causes immune system-related pathology and neoplastic events but no motor neuron defects[END_REF][START_REF] Dejesus-Hernandez | Expanded GGGGCC hexanucleotide repeat in non-coding region of C9ORF72 causes chromosome 9plinked frontotemporal dementia and amyotrophic lateral sclerosis[END_REF][START_REF] Renton | A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD[END_REF].

While only one study reported mild locomotor abnormalities [START_REF] Atanasio | C9orf72 ablation causes immune dysregulation characterized by leukocyte expansion, autoantibody production, and glomerulonephropathy in mice[END_REF], five described C9orf72 KO mice suffering from strong immune system defects, including splenomegaly, massive myeloid and lymphoid cell infiltration in the spleen and the lymph nodes, an elevated rate of autoantibodies, and elevated levels of several pro-inflammatory cytokines in the bloodphenotypes resembling an autoimmune syndrome [START_REF] Atanasio | C9orf72 ablation causes immune dysregulation characterized by leukocyte expansion, autoantibody production, and glomerulonephropathy in mice[END_REF][START_REF] Burberry | Loss-of-function mutations in the C9ORF72 mouse ortholog cause fatal autoimmune disease[END_REF][START_REF] Jiang | Gain of Toxicity from ALS/FTD-Linked Repeat Expansions in C9ORF72 Is Alleviated by Antisense Oligonucleotides Targeting GGGGCC-Containing RNAs[END_REF][START_REF] O'rourke | C9orf72 is required for proper macrophage and microglial function in mice[END_REF][START_REF] Sudria-Lopez | Full ablation of C9orf72 in mice causes immune system-related pathology and neoplastic events but no motor neuron defects[END_REF]. These defects were reported in homozygous but not heterozygous mice. Importantly, ALS patients carry the mutant expansion in only one C9orf72 allele, which leads to some decrease of C9orf72 mRNA and protein levels [20,22*]. In C9orf72 ALS patients, the link between their MN disease and possible immune system dysfunctions is not obvious. Although, an increased number of autoimmune diseases in ALS/FTD patients has been reported, a specific link with C9orf72 remains to be determined [START_REF] Miller | Increased prevalence of autoimmune disease within C9 and FTD / MND cohorts Completing the picture[END_REF]. Microglial cells isolated from C9orf72 KO (but not from C9orf72 +/-) mice displayed a lysosomal transport defect, also noticed in microglial cells of motor cortex and spinal cord postmortem tissues of mutant C9orf72 ALS patients [START_REF] O'rourke | C9orf72 is required for proper macrophage and microglial function in mice[END_REF].

Removing murine C9orf72 from neuronal and glial cell precursors only (but not targeting immune cells, including microglial cells), did not lead to any pathological neuronal signs of ALS or neuroinflammatory reaction [START_REF] Koppers | C9orf72 ablation in mice does not cause motor neuron degeneration or motor deficits[END_REF]. Therefore, the current hypothesis is a mechanism of gain-of-toxic function in neurons (and glial cells of neural origin), possibly potentiated by a downregulation of C9orf72 and a loss-of-function haplo-insufficiency effect in immune cells, which, however, remains to be further characterized in mutant C9orf72 ALS patients. TBK1 (Tank-Binding Kinase-1) discovered in 2015 as a new ALS-linked gene, was previously known for its functions in autophagy but also immune system regulation (originally described as a mediator of NF-κB signalling pathway) [START_REF] Cirulli | Exome sequencing in amyotrophic lateral sclerosis identifies risk genes and pathways[END_REF][START_REF] Freischmidt | Haploinsufficiency of TBK1 causes familial ALS and fronto-temporal dementia[END_REF][START_REF] Pomerantz | NF-kappaB activation by a signaling complex containing TRAF2, TANK and TBK1, a novel IKK-related kinase[END_REF]. Full deletion of Tbk1 in mice on a C57Bl6 genetic background, is embryonic lethal (due to liver failure), but some Tbk1 KO mice on a 129S5 genetic background get born and, interestingly, develop a phenotype very similar to the C9orf72 KO mice, including massive infiltrations of mononuclear cells in different organs, higher rate of circulating monocytes and greater amounts of pro-inflammatory cytokines in the blood [START_REF] Marchlik | Mice lacking Tbk1 activity exhibit immune cell infiltrates in multiple tissues and increased susceptibility to LPS-induced lethality[END_REF]. Mutations in TBK1, discovered in familial ALS cases, result (in most cases) in a loss-of-function of the mutant protein, strongly suggesting a mechanism of haploinsufficiency [START_REF] Cirulli | Exome sequencing in amyotrophic lateral sclerosis identifies risk genes and pathways[END_REF][START_REF] Freischmidt | Haploinsufficiency of TBK1 causes familial ALS and fronto-temporal dementia[END_REF]. In mice, heterozygous deletion of Tbk1, did not lead to any obvious MN degeneration (at least, not until 6 months of age) [29*].

However, and surprisingly, in the neurodegenerative and inflammatory context of mutant SOD1 G93A ALS mice, heterozygous deletion of Tbk1 induced, first, an accelerated MN denervation, followed, second, by decreased microglial activation at late disease stages and, remarkably, an increased survival. This highlights a possible dual role of Tbk1 in MNs and microglial cells, that both can influence the ALS disease course separately and distinctly.

In this context, another gene previously reported for carrying rare (most likely loss-offunction) mutations in ALS, OPTN (encoding for Optineurin), implicated in autophagy and regulation of NF-κB, interacts and gets phosphorylated by TBK1 [START_REF] Maruyama | Mutations of optineurin in amyotrophic lateral sclerosis[END_REF][START_REF] Millecamps | Screening of OPTN in French familial amyotrophic lateral sclerosis[END_REF][START_REF] Zhu | Optineurin Negatively Regulates TNFa-Induced NF-kB Activation by Competing with NEMO for Ubiquitinated RIP[END_REF]. While Optn KO mice did not show a clear MN degeneration, they did display signs of distal axonopathy [START_REF] Ito | RIPK1 mediates axonal degeneration by promoting inflammation and necroptosis in ALS[END_REF].

Optn KO mice also produced elevated amounts of pro-inflammatory cytokines, and transcriptional analysis of Optn KO microglial cells revealed a rather pro-inflammatory phenotype. Therefore, both mutations in Optn and Tbk1 induce inflammatory dysregulations, potentially contributing to ALS.

Other genes expressed by immune cells have been suspected as causative or modifiers of ALS, including CX3CR1, encoding the receptor for the chemokine CX3CL1 (fractalkine), constitutively expressed by neurons including MNs. Microglia are the only CNS cells expressing CX3CR1, making CX3CL1/CX3CR1 a candidate pathway for a MN-microglia cross talk [START_REF] Harrison | Role for neuronally derived fractalkine in mediating interactions between neurons and CX3CR1-expressing microglia[END_REF][START_REF] Jung | Analysis of Fractalkine Receptor CX3CR1 Function by Targeted Deletion and Green Fluorescent Protein Reporter Gene Insertion[END_REF]. Membrane-bound fractalkine is considered a main actor to keep microglia in a homeostatic, non-activated state [START_REF] Cardona | Control of microglial neurotoxicity by the fractalkine receptor[END_REF]. Deletion of Cx3cr1 in SOD1 G93A ALS mice revealed increased MN loss and a tendency to higher microglial activation. SOD1 G93A /Cx3cr1 -/-males (but not females) displayed a faster decline in motor functions and a reduced survival compared to SOD1 G93A /Cx3cr1 +/-males [36,37*]. In ALS patients, specific variants in the CX3CR1 gene have been associated with a faster progression of the disease, although there was no association with ALS disease risk [38,

Lymphocytes implicated in ALS neurodegeneration and a possible biomarker in ALS

Originally, CD4+ cells were the main T cell population thought to be implicated in ALS, with removal of CD4+ cells (CD4 KO mice) showing reduced survival of SOD1 G93A ALS mice [START_REF] Beers | CD4+ T cells support glial neuroprotection, slow disease progression, and modify glial morphology in an animal model of inherited ALS[END_REF][START_REF] Chiu | T lymphocytes potentiate endogenous neuroprotective inflammation in a mouse model of ALS[END_REF]. Simplified, CD4+ cells can be subdivided in regulatory (Treg) Interestingly, in CD8a -/-/SOD1 G93A mice, MNs survived better in the lumbar spinal cord, while in ß2M -/-/SOD1 G93A mice, it was not the case, however MNs were protected in the cervical spinal cord [13**,14**,51]. Since CD8+ T cell toxicity towards MNs was dependent on MHC-I recognition by CD8+ T cells in vitro, a compensatory mechanism could explain the difference obtained in ß2M -/-and CD8a -/-mice and the heterogeneity between cervical and lumbar MNs in ß2M -/-mice [13**,14**]. As MHC-I is expressed also by myeloid cells, an additional effect of deleting MHC-I could also come from deregulated microglia/macrophage reactivity. In addition, CD8+ T cells seem to have a dual role in ALS, with a protective effect at the periphery (accelerated muscle atrophy and axonal defects in ß2M -/-/SOD1 G93A mice), and a toxic role on MN survival when infiltrating the CNS [13**,14**] (Fig1). Of note, for both CD4+ and CD8+ cells, it remains to be determined if their role in ALS disease is linked to mutant gene expression (e.g. mutant SOD1) or their neuroinflammatory responses. Another subpopulation of lymphocytes (which are part, however, of the innate immunity, as they do not recognize specific antigens), the Natural killer (NK) cells with their cytotoxic activity, have recently also been studied in ALS. A longitudinal study showed an increased number of NK cells in the blood of ALS patients compared to controls, but also in ALS patients along the disease [52*]. Interestingly, infiltrating NK cells have been observed in the spinal cord of ALS mice, however, decreasing the number of NK cells in SOD1 G93A mice had no impact on disease [11,50**].

Mast cell involvement in ALS

Mast cells, granulated leucocytes involved in allergic reactions, have recently gained interest as the target cells of Masitinib (AB1010), a Tyrosine Kinase inhibitor, tested in clinical trials on ALS patients (ClinicalTrials.gov NCT02588677). Mast cells were observed in muscles (extensor digitorum longus) and sciatic nerves of symptomatic SOD1 G93A ALS rats but not in the spinal cord. Attention has therefore been focused on their impact at the periphery [53**,54*]. Presence of mast cells (in variable amounts compared to controls), was also described in ALS patient quadriceps muscles with frequent degranulation, suggesting mast cell reaction and a potential role in ALS [54*]. In SOD1 G93A rats, Masitinib treatment led to a decreased number of mast cells in muscles and sciatic nerves and delayed neuromuscular junction denervation, suggesting a deleterious role for mast cells towards axonal integrity in ALS [54*,55] (Fig. 1). However, Masitinib is also an inhibitor of Csf1-R, required for macrophage and microglia survival. Since selective inhibition of the Csf1-R (with GW2580) was also potent at slowing the disease course in SOD1 G93A mice [56], the impact of Masitinib in ALS could be through mast cells and macrophage/microglial cells, and specific depletion or inhibition of mast cells would be needed to definitely conclude on mast cell contribution in ALS.

Deregulation of monocytes in ALS

For their easy accessibility in the blood, and since, as part of the myeloid lineage, they share common functions with microglial cells, monocytes have gained recent interest in ALS.

While analysis of overall proportions of monocytes among all the leucocytes was not sensitive enough to show differences between ALS patients and healthy controls, a recent longitudinal study, performed over 3 years, highlighted an increased monocyte number (and also of the whole leucocyte content in the blood) during the course of ALS, although it was

Conclusion

Although, MNs are the primary cells degenerating in ALS, rising evidence show that ALS has an inflammatory component, with some ALS related genes linked to different immune functions. Defects caused by mutations in those genes are likely to drive defects in their immune functions, potentially impacting ALS disease course. Modifying immune cells through bone marrow transplantation has been envisaged and targeting specific myeloid or lymphoid populations is the focus of ongoing clinical trials. However, better knowledge of the specific deregulations of the different subpopulations of immune cells (including CD4+ Tregs, CD8+ T cells, mast cells and monocytes) and this, over the course of the disease, between different ALS patient cohorts, are now necessary and the essential next step required for the development of immune-related therapies and biomarkers in ALS.

Key points:

• Recent genes discovered in ALS reinforce the link between the disease and alteration of the immune system.

• Blood lymphocytes and in particular regulatory T lymphocytes appear as promising predictors of the rate of disease progression.

• Presence of mast cells in muscles in ALS models and patients and deregulated monocytes in ALS patients highlight new immune cell players at the periphery to consider for future therapy.

• Failure of fundamental functions of monocytes calls on a possible systemic immune deregulation in ALS

  39*]. TREM2, well-known for its mutations causing rare forms of Alzheimer's disease, encodes for Triggering Receptor Expressed Myeloid cell 2, expressed by myeloid cells and in particular microglial cells [40]. A specific variant in TREM2 (p.R47H) was identified as a risk factor for ALS without, however, being correlated with the age of onset, the site of the first symptoms or disease progression [41]. TREM2 encodes for an extracellular receptor involved in immune functions, especially phagocytosis. The p.R47H variant was associated with maturation defects of the receptor causing impaired phagocytosis [42]. Increased expression of Trem2 mRNA was measured in the spinal cord of symptomatic mutant SOD1 G93A mice, but also sporadic ALS patients [41]. Very recently, upregulation of Trem2 mRNA was measured, at early disease stages in ALS mice, with a new technique of spatial transcriptomics, allowing full transcriptome analysis directly on tissue sections [43**]. Interestingly, this study revealed that Tyrobp, which forms a complex with Trem2 that triggers phagocytosis, was up-regulated on the mRNA level, even before Trem2. In addition, the Trem2-ApoE pathway (Trem2 is also known for inducing ApoE signalling) was previously shown to trigger a common molecular signature of microglial cells in several neurodegenerative conditions, including ALS [44**]. Deletion of Trem2 in mutant SOD1-expressing microglial cells, induced downregulation of inflammatory genes and restored microglial homeostatic gene expression [44**]. The example of Trem2 is an interesting indicator of microglial implications in ALS, suggesting that such implication could be triggered both by internal defects, e.g. caused by genetic ALSlinked mutations (as SOD1), but also by (perturbed) external signalling, mediated, through the neurodegenerative/neuroinflammatory context.

  and effector (Teff) T cells, with Tregs regulating the proliferation of Teff cells. In SOD1 G93A ALS mice, passive transfer of T cell populations enriched in Tregs compared to whole CD4+ T cells, led to a more pronounced increase in survival [45,46]. Treg expansion in SOD1 G93A mice (using peripheral injection of Interleukin 2 (IL-2)/IL2 antibody complex), decreased microglial activation and increased MN and mouse survival [47**]. In ALS patients, a higher proportion of Treg in the blood was correlated with a slower rate of disease progression. [46-48]. Functional assays with Tregs of ALS patients showed their reduced ability to suppress the Teff proliferation, compared to control Tregs [49**]. Therefore, both reduced numbers and impaired suppressive function could influence ALS progression. A phase II clinical study assessing the potential of IL-2 as a therapeutic agent is currently in progress in ALS (ClinicalTrials.gov NCT03039673). FoxP3, a marker of Treg was found downregulated in ALS patients' Tregs compared to controls. FoxP3 levels were also inversely correlated with the rate of disease progression, with FoxP3 expression being reduced in rapidly progressing patients [48,49**]. Importantly, a prospective study pointed out low levels of FoxP3, not only reflecting a rapid disease progression at the time of blood collection, but also able to predict future rapid disease progression rate. Therefore, FoxP3 appears as a promising biomarker for ALS disease progression [48].More recent studies have focused on the, so far in ALS understudied, CD8+ lymphocytes.While previous reports mentioned a very late infiltration of CD8+ T cells (compared to CD4+ cells) in the spinal cord of SOD1 G93A ALS mice (arguing in favor of no possible impact of these cells), two recent studies measured the presence of infiltrating CD8+ T cells as early as disease onset [10,13**,14**]. To study CD8+ T cell involvement, ALS mice either lacking functional CD8+ T cells (CD8a -/-/SOD1 G93A ) or defective for CD8+ T cell actions (ß2microglobulin (ß2M) -/-/SOD1 G93A , displaying no MHC-I expression) were generated [13**,14**] or were injected with anti-CD8 antibodies [50**]. Although survival of CD8a -/- /SOD1 G93A mice or of SOD1 mice injected with anti-CD8, was not modified, survival of ß2M - /-/SOD1 G93A mice showed a different outcome in two independent studies with either, for the earlier study, no modified onset but a decreased survival[51], or in the more recent study, an earlier onset (measured by muscle weakness) followed by a prolonged survival [13**].

  not correlated with the functional ALSFRS-R score [52*,57]. Expression of the specific marker, CX3CR1, in this longitudinal study, correlated, however, with the ALS Functional Rating Scale Revised (ALSFRS-R) score [52*]. Monocytes can be divided into two major populations, classical monocytes, considered to be inflammatory and recruited into the tissues upon inflammation, and non-classical or patrolling monocytes, in charge of tissue homeostasis. A higher proportion of classical to non-classical monocytes has been described in ALS patients compared to healthy controls [58*]. One hypothesis would therefore be, that deregulated proportions of monocyte subtypes could lead to dysfunctions of immune responses, contributing to ALS disease. However, possible intrinsic dysfunctions in ALS monocytes have also been reported, and included alterations in phagocytosis, cytokine secretions upon external stimuli, and of their transcriptome [57,58*,59**] (Fig. 1). Upregulation of genes involved in inflammatory responses such as Interleukine-1ß, Interleukine-8 or NLRP3, and downregulation of genes involved in Tumor Necrosis Factor or Toll Like Receptor signaling (two important pathways of the innate inflammatory response), were revealed with RNAseq analyses in monocytes from ALS patients compared to controls, suggesting a possible dysfunction of myeloid cells in ALS [57,59**].

[ 39 ]

 39 Calvo A, Moglia C, Canosa A, et al. Common polymorphisms of chemokine (C-X3-C motif) receptor 1 gene modify amyotrophic lateral sclerosis outcome: A populationbased study. Muscle and Nerve 2018;57:212-6. * This study indicates that a specific variant in CX3CR1 is associated with a faster disease progression in ALS patients. [40] Sims R, Van der Lee SJ, Naj AC, et al. Rare coding variants in PLCG2, ABI3, and TREM2 implicate microglial-mediated innate immunity in Alzheimer's disease. Nat Genet 2017;49:1373-87. [41] Cady J, Koval ED, Benitez BA, et al. The TREM2 variant p.R47H is a risk factor for sporadic amyotrophic lateral sclerosis Janet. JAMA Neurol 2014;71:449-53. [42] Kleinberger G, Yamanishi Y, Suárez-calvet M, et al. TREM2 mutations implicated in neurodegeneration impair cell surface transport and phagocytosis. Sci Transl Med 2014;6. [43] Maniatis S, Vickovic S, Braine C, et al. Spatiotemporal dynamics of molecular pathology in amyotrophic lateral sclerosis. Science 2019;93:89-93. ** This study provides a spatial transcriptomic analysis of SOD1 G93A mouse spinal cord over the course of the disease, and identifies dynamic regulations of several immune signalling pathways. [44] Krasemann S, Madore C, Cialic R, et al. The TREM2-APOE pathway drives the transcriptional phenotype of dysfunctional microglia in neurodegenerative diseases. Immunity 2017;47:566-81. ** This study identifies a molecular signature of microglial cells common to several neurological disorders and shows how the Trem2-ApoE pathway mediates a switch towards this reactive phenotype, which then could be targetted to reduce deletrious microglial cell reactivity. [45] Banerjee R, Mosley RL, Reynolds AD, et al. Adaptive immune neuroprotection in G93A-SOD1 amyotrophic lateral sclerosis mice. PLoS One 2008;3:e2740. [46] Beers DR, Henkel JS, Zhao W, et al. Endogenous regulatory T lymphocytes ameliorate amyotrophic lateral sclerosis in mice and correlate with disease progression in patients with amyotrophic lateral sclerosis. Brain 2011;134:1293-314. [47] Sheean RK, McKay FC, Cretney E, et al. Association of regulatory T-Cell Expansion with progression of amyotrophic lateral sclerosis a study of humans and a transgenic mouse model. JAMA Neurol 2018;75:681-9. ** This study shows a correlation between regulatory T cell (Treg) levels and rate of disease progression in ALS patients and reports a protective effect of IL2 treatment inducing Treg expansion in SOD1 G93A ALS mice. [48] Henkel JS, Beers DR, Wen S, et al. Regulatory T-lymphocytes mediate amyotrophic lateral sclerosis progression and survival. EMBO Mol Med 2013;5:64-79. [49] Beers DR, Zhao W, Wang J, et al. ALS patients' regulatory T lymphocytes are dysfunctional, and correlate with disease progression rate and severity. Jci 2017;2:e89530. ** This study demonstrates how ALS patient regulatory T cells have defective suppressive functions (on the effector T cell proliferation) and shows an association between the level of failure and the rate of disease progression. This indicates that restoring their function could be effective in slowing disease progression. [50] Komine O, Yamashita H, Fujimori-Tonou N, et al. Innate immune adaptor TRIF deficiency accelerates disease progression of ALS mice with accumulation of aberrantly activated astrocytes. Cell Death Differ 2018:1-17. ** First evaluation of the possible involvement of Natural Killer cells in ALS. [51] Staats KA, Schönefeldt S, Van Rillaer M, et al. Beta-2 microglobulin is important for disease progression in a murine model for amyotrophic lateral sclerosis. Front Cell Neurosci 2013;7:6-10. [52] Murdock BJ, Zhou T, Kashlan SR, et al. Correlation of peripheral immunity with rapid Amyotrophic lateral sclerosis progression. JAMA Neurol 2017;74:1446-54. * A longitudinal study aiming to correlate disease progression and long-term fluctuations of blood cell numbers and markers in the blood of ALS patients. [53] Trias E, Ibarburu S, Barreto-Núñez R, et al. Evidence for mast cells contributing to neuromuscular pathology in an inherited model of ALS. JCI Insight 2017;2:1-14. ** This study documents how mast cells could contribute to the deregulation of the neuromuscular unit in the mutant SOD1 ALS rat model and proposes mast cells as a novel potential target to improve ALS disease. [54] Trias E, King PH, Si Y, et al. Mast cells and neutrophils mediate peripheral motor pathway degeneration in ALS Graphical abstract Find the latest version : Mast cells and neutrophils mediate peripheral motor pathway degeneration in ALS. J Clin Investig Insight 2018;3. * Further evidence of the peripheral effect of mast cells and neutrophils in ALS, with a focus on ALS patient tissues [55] Trias E, Ibarburu S, Barreto-Núñez R, et al. Post-paralysis tyrosine kinase inhibition with masitinib abrogates neuroinflammation and slows disease progression in inherited amyotrophic lateral sclerosis. J Neuroinflammation 2016;13:177. [56] Martínez-Muriana A, Mancuso R, Francos-Quijorna I, et al. CSF1R blockade slows the progression of amyotrophic lateral sclerosis by reducing microgliosis and invasion of macrophages into peripheral nerves. Sci Rep 2016;6:1-13. [57] Zondler L, Müller K, Khalaji S, et al. Peripheral monocytes are functionally altered and invade the CNS in ALS patients. Acta Neuropathol 2016;132:391-411. [58] Zondler L, Feiler MS, Freischmidt A, et al. Impaired activation of ALS monocytes by exosomes. Immunol Cell Biol 2017;95:207-14. * This study provides a functional analysis of ALS patient monocytes showing defects in essential functions and explores if exosomes are important mediators of immune dysregulations of monocytes in ALS. [59] Zhao W, Beers DR, Hooten KG, et al. Characterization of gene expression phenotype in amyotrophic lateral sclerosis monocytes. JAMA Neurol 2017;74:677-85.** This transcriptomic study shows that monocytes from ALS patients have a more "proinflammatory" profile, particularly in fast progressing ALS patients.

Figure 1 .

 1 Figure 1. Evidence of involvement of neuroinflammatory processes in amyotrophic lateral sclerosis (ALS). New ALS-linked genes with important immune functions are shown in boxes. Different subpopulations of immune cells that have been linked to ALS: CD4+ (including Foxp3+ regulatory cells) and CD8+ T lymphocytes, mast cells, monocytes and microglial cells are depicted with their described functions in the different tissues.

Financial support and sponsorship

This work was supported by Thierry Latran Foundation, EraNet Neuron, ALSA, NRJ-Institut de France, ARMC, SLAFR, La Longue route, AC was financed by the French ministry of higher education, research and innovation, and ARSLA.

Conflicts of interest

The authors declare no conflict of interest


Adrien Barton

Christina Khnaisser

Luc Lavoie

Jean-François Ethier

Ambiguities in Medical Bitemporalized Relational Databases: a Referent Tracking View

Keywords: Temporalized relational database, Referent tracking, Information ambiguity, Valid time, Transaction time Temporalized relational database, Referent tracking, Information ambiguity, Valid time, Transaction time

An analysis based on referent tracking systems shows that a classical medical application of bitemporalized relational database that uses classes as attribute values is ambiguous in several respects. This suggests that to avoid such ambiguities, bitemporalized relational databases could be structured on the basis of ontological representations that give adequate attention to particulars.

1.!Introduction

Temporalized relational databases ("T-RDB" for short) are used in the medical domain to describe the temporal dimensions of relevant medical entities. To be able to exchange reliably the information stored in T-RDB with other information systems, T-RDB should be structured non-ambiguously. However, this article will show that a classical medical application of T-RDB can be ambiguous in several respects, because of its use of classes as values in an attribute. The analysis will rest on the referent tracking paradigm [START_REF] Ceusters | Strategies for referent tracking in electronic health records[END_REF] that uses biomedical ontologies in the description of particular entities such as a given health care professional, a given patient, his diseases or pathological processes, as well as the relevant temporal dimensions.

2.!Temporalized relational databases

2.1.!Valid time and agent-relativization

Consider the following scenario (called "scenario A"), inspired by [START_REF] Combi | Temporal Information Systems in Medicine[END_REF]: on April 1 st 2017, Mr. Hubbard conveys to Dr. Jones that after coming back from a trip to India in January, he had high fever and nausea from February 3 rd to February 19 th ; Dr. Jones enters into his database his diagnosis that Hubbard had malaria during that time. On May 17 th , in

Introduction

Temporalized relational databases ("T-RDB" for short) are used in the medical domain to describe the temporal dimensions of relevant medical entities. To be able to exchange reliably the information stored in T-RDB with other information systems, T-RDB should be structured non-ambiguously. However, this article will show that a classical medical application of T-RDB can be ambiguous in several respects, because of its use of classes as values in an attribute. The analysis will rest on the referent tracking paradigm [START_REF] Ceusters | Strategies for referent tracking in electronic health records[END_REF] that uses biomedical ontologies in the description of particular entities such as a given health care professional, a given patient, his diseases or pathological processes, as well as the relevant temporal dimensions.

Temporalized relational databases

Valid time and agent-relativization

Consider the following scenario (called "scenario A"), inspired by [START_REF] Combi | Temporal Information Systems in Medicine[END_REF]: on April 1 st 2017, light of medical tests and a finer estimation of when the symptoms stopped, Dr. Jones diagnoses that Hubbard had dengue fever from February 3 rd to February 24 th ; he corrects immediately the database.

Suppose that Jones was using a T-RDB with the valid-time relvar [3] R V = <PAT, DIS, @V, AG>, that represents the following predicate p V (PAT,DIS,@V,AG):

'(PAT is a particular patient) and (DIS is a class of diseases) and (@V is a time) and (AG is a particular agent) and [AG currently believes the following proposition: (PAT has a disease of class DIS during @V)]' where @V is called a "valid time". Following the realist methodology [START_REF] Smith | Ontological realism: A methodology for coordinated evolution of scientific ontologies[END_REF], this predicate distinguishes clearly the particulars from the universals or classes ("a time" refers to any mereological sum [START_REF] Varzi | Mereology[END_REF] of particular time instants and intervals). Note that here, the values of the attribute DIS are classes: this will be the cause of the problems mentioned later. On April 1 st , the relation R V includes the tuple tp 1 V = (Hubbard, Malaria, [Feb3:Feb19], Jones) (dropping the reference to 2017). On May 17 th , tp 1

V is replaced by tp 2 V = (Hubbard, Dengue, [Feb3:Feb24], Jones).

2.2.!Transaction time

It is important to keep track of those tuple modifications in the T-RDB for audit purposes. Therefore, the database may include the "log" [START_REF] Date | Time and relational theory: temporal databases in the relational model and SQL[END_REF] of relation R V , which is itself a relvar named here "R V, T ": a bitemporal relvar including both valid times and transaction times, stating when some tuple was present in the relation R V . Its value includes the following tuples tp 1 V,T and tp 2 V,T on May 17 th :

PAT DIS @V AG @T tp 1 V,T Hubbard Malaria [Feb3:Feb19] Jones [Apr1:May16] tp 2 V,T Hubbard Dengue [Feb3:Feb24] Jones [May17:ufn]
where @T is called a "transaction time" and "ufn" stands for "until further notice", meaning that the described tuple is still presently unchanged in R V . A database system using valid time and transaction time is called a "bitemporalized RDB" ("B-RDB"). tp 1 V,T means that according to R V , Jones believed only from April 1 st to May 16 th (because of the closed-world assumption) that Hubbard had malaria during [Feb3:Feb19]; this implies the propositions: (S 1a ) "Jones asserts on Apr1 that Hubbard had malaria during exactly [Feb3:Feb19]." and (S 1b ) "Jones asserts on May17 that Hubbard did not have malaria during exactly [Feb3:Feb19]." Similarly, tp 2 V,T implies: (S 2 ) "Jones asserts on May17 that Hubbard had dengue during exactly [Feb3:Feb24]." We will see later that (S 1a ), (S 1b ) and (S 2 ) are ambiguous in several respects.

3.!Bridge with referent-tracking systems

3.1.!IUI repository and referent-tracking database

A referent tracking (RT) system is composed of two parts [START_REF] Ceusters | Strategies for referent tracking in electronic health records[END_REF]. First, an IUI (Instance Unique Identifier) repository, which is an inventory of identifiers for individual entities, such as a specific patient Mr. Williams, his heart, his atrial fibrillation disease, and each of his atrial fibrillation episode. Second, the referent-tracking database ("RT-DB"), which is an inventory of assertions concerning the relationships between particulars, as well as between particulars and universals, and the ways those change over time. In the following, we write (as in [START_REF] Ceusters | Strategies for referent tracking in electronic health records[END_REF]) "IUI A " for referring to the particular unique identifier IUI A , and "#IUI A " for referring to the entity referred to by IUI A . For example, if IUI Jones is the IUI referring to Jones, then #IUI Jones is Jones. In an ontological context, we will also write in bold the names of particulars and relations involving at least a particular, and use italic for universals.

3.2.!Bridging B-RDB with RT-DB

An RT-DB can include a variety of kind of tuples [START_REF] Ceusters | Strategies for referent tracking in electronic health records[END_REF][START_REF] Ceusters | Negative findings in electronic health records and biomedical ontologies: a realist approach[END_REF], such as: PtoP tuples that each state a relation between particulars; PtoU tuples that each state an instantiation of a universal by a particular; and PtoLackU tuples that each state the lack of instantiation of a universal by a particular. Let's introduce IUI disease_H as referring to the disease that caused Hubbard's February symptoms of fever and nausea. (S 1a ) (as defined above in section 2.2) can be expressed by a combination of two RT tuples:

•! PtoP 1 = < IUI Jones , Apr1, inheres_in, RO, (IUI disease_H ,IUI Hubbard ), [Feb3:Feb19] >, which describes that Jones asserts on Apr1 that #IUI disease_H inheres_in Hubbard during [Feb3:Feb19] (where inheres_in is a relation of RO, the Relation Ontology [START_REF] Smith | Relations in biomedical ontologies[END_REF]). •! PtoU 1 = < IUI Jones , Apr1, inst, DO, IUI disease_H , Malaria, [Feb3:Feb19] >, which describes that Jones asserts on Apr1 that #IUI disease_H instance_of Malaria during [Feb3:Feb19] (where Malaria is a class of DO, the Disease Ontology). Altogether, those two tuples describe that Jones asserts on Apr1 that there is an instance of Malaria (namely, #IUI disease_H ) inhering in Hubbard during [Feb3:Feb19]. We will now show ambiguities in the B-RDB tuples by describing the situation with RT-tuples.

4.!The ambiguities of T-RDBs

4.1.!Ambiguity 1: Having an asymptomatic disease vs. not having a disease

A first ambiguity is revealed when trying to express (S 1b ) by RT tuples. (S 1b ) can mean that Jones stated on May17 that #IUI disease_H was not an instance of Malaria, in which case it is synonymous with the RT tuple PtoLackU 1 = < IUI Jones , May17, identical_with, RO, IUI disease_H , Malaria, [Feb3:Feb19] > (which describes that Jones asserted on May17 that there is no instance of Malaria that is identical_with #IUI disease_H ). Alternatively, (S 1b ) can mean that Jones stated on May17 that Hubbard did not have any instance of malaria during [Feb3:Feb19], in which case it is synonymous with the RT tuple PtoLackU 2 = < IUI Jones , May17, inheres_in, RO, IUI Hubbard , Malaria, [Feb3:Feb19] >. PtoLackU 2 asserts a stronger statement than the one asserted by PtoLackU 1 , as the former logically implies the latter. The difference between PtoLackU 1 and PtoLackU 2 is medically relevant: if Hubbard suffered during [Feb3:Feb19] from an asymptomatic malaria while having at the same time a dengue fever that caused his symptoms of fever and nausea, PtoLackU 1 would hold, but not PtoLackU 2 ; on the other hand, if Hubbard only had dengue fever during [Feb3:Feb19] and no malaria, PtoLackU 2 would hold. But it is ambiguous whether (S 1b ) means PtoLackU 1 or PtoLackU 2 2 .

4.2.!Ambiguity 2: Re-categorization of a formerly considered disease vs. consideration of a new disease

A second ambiguity is revealed when trying to express the B-RDB tuple tp 2 V,T =(Hubbard, Dengue, [Feb3:Feb24], Jones, [May17:ufn]) with RT tuples. In the case at hand, IUI disease_H had been defined as the IUI referring to Hubbard's disease that caused his symptoms of high fever and nausea in February 3 . Then tp 2 V,T describes that Jones stated on May17 two things about #IUI disease_H :

•! it inhered in Hubbard from Feb3 to Feb24, as expressed by PtoP 2 = < IUI Jones ; May17 ; inheres_in ; RO ; (IUI disease_H ,IUI Hubbard ) ; [Feb3:Feb24] > •! it was an instance of Dengue, as expressed by PtoU 2 = < IUI Jones ; May17 ; inst ; DO ; IUI disease_H ; Dengue; [Feb3:Feb24] > Suppose now that we are not in "scenario A", but in a "scenario B", that differs from scenario A in two respects. First, Jones learns on May17 that Hubbard's description of his February symptoms of high fever and nausea on April 1 st were a lie (or a joke) -he never had them, and thus never had malaria nor dengue in February; second, he learns on May 17 that Hubbard had during [Feb3:Feb24] an unrelated hyperthyroidism. Instead of writing tp 2 V,T , Jones would introduce in the log of his B-RDB the tuple tp 3 V,T =(Hubbard, Hyperthyroidism, [Feb3:Feb24], Jones, [May17:ufn]).

In such a case, we should not use IUI disease_H to refer to Hubbard's hyperthyroidism, as the latter is unrelated to the fever and nausea symptoms Hubbard allegedly had in February. Instead, IUI disease_H would be an IUI without a reference 4 ; and we would create a new IUI (let's say IUI disease_H_2 ) to refer to Hubbard's hyperthyroidism, such that Jones states on May17 two things about #IUI disease_H_2 :

•! it inhered in Hubbard from Feb3 to Feb24, as expressed by PtoP 3 = < IUI Jones ; May17 ; inheres_in ; RO ; (IUI disease_H_2 ,IUI Hubbard ) ; [Feb3:Feb24] > •! it was an instance of Hyperthyroidism, as expressed by PtoU 3 = < IUI Jones ; May17 inst ; DO ; IUI disease_H_2 ; Hyperthyroidism ; [Feb3:Feb24] > Note that the B-RDB descriptions of scenarios A and B are similar (the only difference between tp 3 V,T and tp 2 V,T is the use of "Hyperthyroidism" instead of "Dengue"); but this does not describe an important difference between scenarios A and B, revealed by the RT descriptions of both scenarios: the RT description of scenario A uses the formerly introduced IUI disease_H (with PtoP 2 and PtoU 2 ), whereas the RT description of scenario B uses the newly introduced IUI disease_H_2 (with PtoP 3 and PtoU 3 ).

4.3.!Ambiguity 3: One vs. several pathological processes

A third ambiguity concerns the number of entities. Consider the following relation R PP,V (with "DIS" replaced by "PP", referring to "pathological process") :

PAT PP @V Williams
AF episode [Jun6:Jun15] Note first that the proposition associated to this tuple cannot be 'Williams had an AF episode that spanned exactly the time interval [Jun6:Jun15]', as this describes a so-called "telic" fact that cannot be represented in point-based semantics [START_REF] Terenziani | Reconciling point-based and interval-based semantics in temporal relational databases: A treatment of the telic/atelic distinction[END_REF]. The semantic of such predicate would rather be 'Williams had one or several AF episodes that together spanned a time interval including [Jun6:Jun15]'. Therefore, Williams may have had only one AF episode spanning the whole interval [Jun6:Jun15]. Alternatively, he may have had two AF episodes: a first one during [Jun6:Jun10], and a second one during [Jun11:Jun15]. There is thus an ambiguity concerning how many AF episodes Williams had. Note that such ambiguity would not appear in a RT system: each AF episode would have its own IUI, therefore the RT data would explicitly describe if Williams had one or several AF episode(s) during the interval [Jun6:Jun15].

5.!Conclusion

This article showed several ambiguities concerning the individual entities underlying the predicates expressed by T-RDBs when the values of some of their attributes (such as DIS in R V or PP in R PP,V ) refer to classes or universals, rather than to individuals; this makes it a particular case of a difficulty named the "assumption of inherent classification" [11,[START_REF] Smith | Relations in biomedical ontologies[END_REF]. This suggests that to avoid such ambiguities, medical T-RDBs could be structured on the basis of ontology-based representations that deal carefully with the particulars involved, such as referent tracking systems.

Corresponding Authors; E-mail: ethierj@gmail.com; adrien.barton@gmail.com. AB acknowledges financial support by the "bourse de fellowship du département de médecine de l'université de Sherbrooke" and the CIHR-funded "Quebec SPOR Support Unit".

Note that in both cases, an additional RT-tuple should be introduced, stating that the universal-tuple PtoU1 above was wrong -see[START_REF] Ceusters | Dealing with mistakes in a referent tracking system[END_REF] for a suggestion of how to represent this as a D-tuple.This example raises a more general issue, namely how we define the identity of a particular disease. In the case at hand, the particular #IUIdisease_H is defined as the disease causing some symptoms of Hubbard (high fever, nausea) during an approximate time period (during the month of February).[START_REF] Smith | Ontological realism: A methodology for coordinated evolution of scientific ontologies[END_REF] This mistake is named "(A1)" in[START_REF] Ceusters | Dealing with mistakes in a referent tracking system[END_REF].