Research Article High-dose intravenous immunoglobulins might modulate inflammation in COVID-19 patients Marı́a Luisa Rodrı́guez de la Concepción1,* , Erola Ainsua-Enrich1,* , Esteban Reynaga2,* , Carlos Ávila-Nieto1, Jose Ramón Santos2, Silvia Roure2, Lourdes Mateu2,4,5, Roger Paredes1,2, Jordi Puig2, Juan Manuel Jimenez2, Nuria Izquierdo-Useros1 , Bonaventura Clotet1,2,3,4,5, Marı́a Luisa Pedro-Botet2,4,5 , Jorge Carrillo1 The use of high-dose of intravenous immunoglobulins (IVIGs) as intensive care unit (China CDC Weekly, 2020; European Center for Disease immunomodulators for the treatment of COVID-19–affected indi- Prevention and Control, 2020). Severe COVID-19 is characterized by viduals has shown promising results. IVIG reduced inflammation in pneumonia, dyspnea, hypoxemia, hyperinflammation, and lymphopenia. these patients, who progressively restored respiratory function. It can rapidly progress to respiratory failure and acute respiratory However, little is known about how they may modulate immune distress syndrome, which is associated with a higher risk of death (Yang responses in COVID-19 individuals. Here, we have analyzed the et al, 2020). An early and sustained increase in cytokine and chemokine levels of 41 inflammatory biomarkers in plasma samples obtained levels has been associated with severe cases of the disease (Huang et al, at day 0 (pretreatment initiation), 3, 7, and 14 from five hospitalized 2020; Lucas et al, 2020). Furthermore, the myeloid cell compartment is COVID-19 patients treated with a 5-d course of 400 mg/kg/d of IVIG. also highly deregulated in those patients, in which a high frequency of The plasmatic levels of several cytokines (Tumor Necrosis Factor, dysfunctional immature neutrophils with an immunosuppressive profile IL-10, IL-5, and IL-7), chemokines (macrophage inflammatory and a reduction of the nonclassical monocytes has been reported protein-1α), growth/tissue repairing factors (hepatic growth fac- (Schulte-Schrepping et al, 2020; Silvin et al, 2020). Thus, it has been tor), complement activation (C5a), and intestinal damage such as postulated that the hyperinflammatory response induced by SARS-CoV- Fatty acid–binding protein 2 and LPS-binding protein showed a 2 does not only induce a dysregulated response of innate immune cells progressive decreasing trend during the next 2 wk after treatment but may also be behind the tissue damage observed in severe COVID-19 initiation. This trend was not observed in IVIG-untreated COVID-19 patients (Merad & Martin, 2020). Autopsy studies have shown a strong patients. Thus, the administration of high-dose IVIG to hospitalized immune infiltration into the lung in deceased severe COVID-19 indi- COVID-19 patients may improve their clinical evolution by modu- viduals that can be categorized in two states of disease progression. The lating their hyperinflammatory and immunosuppressive status. first one is characterized by an accumulation of innate cells associated DOI 10.26508/lsa.202001009 | Received 30 December 2020 | Revised 8 July with a high expression of interferon-related genes (Nienhold et al, 2020). 2021 | Accepted 12 July 2021 | Published online 28 July 2021 In the second one, tissue infiltrate composed by CD8+ T cells and macrophages was associated with a lower expression of interferon- related genes, local complement synthesis (C1q) and complement de- Introduction position (C3b and C5b-9), T-cell exhaustion, and extensive tissue damage (Nienhold et al, 2020). Coronavirus infectious disease 2019 (COVID-19) is a new illness caused Among the treatments assayed for COVID-19, the use of im- by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a munomodulators has shown promising results. Dexamethasone novel coronavirus strain that was identified in China at the beginning of and IL-6 blocking agents have exhibited their usefulness and are 2020 (Wu et al, 2020; Zhu et al, 2020). At present, more than 175 million currently used in the treatment of severe COVID-19–affected indi- of infections and 3.8 million of deaths have been reported globally viduals (The RECOVERY Collaborative Group et al, 2021; The REMAP- (World Health Organization Coronavirus Disease [COVID-19] Dashboard; CAP, 2021). Moreover, other treatments based on anti-complement https://covid19.who.int/). Although the majority of infected individuals drugs are still under clinical evaluation with encouraging results develop mild symptoms, about 10–30% can develop a severe disease (AMY-101, vilobelimab, and eculizumab) (Polycarpou et al, 2020; Kurtovic that requires hospitalization and, eventually, the admission into the & Beeson, 2021). 1 IrsiCaixa AIDS Research Institute, Germans Trias i Pujol Research Institute (IGTP), Can Ruti Campus, Badalona, Spain 2Infectious Diseases Department, Fight Against AIDS Foundation (FLS), Germans Trias i Pujol Hospital, Badalona, Spain 3Chair in Infectious Diseases and Immunity, Centre for Health and Social Care Research (CESS), Faculty of Medicine. University of Vic–Central University of Catalonia (UVic–UCC), Vic, Spain 4Universitat Autonoma de Barcelona, Cerdanyola Del Vallès, Spain 5CIBERes: Centro de investigaciones en Red de Enfermedades Respiratorias Del Instituto Carlos III, Madrid, Spain Correspondence: jcarrillo@irsicaixa.es; mlpbotet.germanstrias@gencat.cat *Marı́a Luisa Rodrı́guez de la Concepción, Erola Ainsua-Enrich, and Esteban Reynaga contributed equally to this work © 2021 Rodrı́guez de la Concepción et al. https://doi.org/10.26508/lsa.202001009 vol 4 | no 9 | e202001009 1 of 7 Intravenous immunoglobulins (IVIGs) are intensively used for Results and Discussion the treatment of immunodeficient individuals as “replacement therapy.” However, at high dose (1–2 g/kg), they are also used as Previously, we have shown that the administration of high-dose of “immunomodulatory agents” for some autoimmune diseases. IVIG IVIG to five COVID-19 patients was well tolerated and improved their can reduce inflammation by several mechanisms which include recovery, normalizing the hyperinflammation status and restoring the development of T-regulatory cells, the reduction of autoan- the levels of lymphocytes (Reynaga et al, 2020). At the start of tibodies and cytokines levels, the prevention of the activation of treatment, they all showed a PaO2/FiO2 values ≤300 mmHg and innate immune cells, or by blocking the activation of the com- multilobar pneumonia and were receiving supplemental oxygen plement cascade (Schwab & Nimmerjahn, 2013). We and others therapy. All these clinical parameters improved notoriously after have shown the success in the treatment of COVID-19 using high treatment initiation (Reynaga et al, 2020). Here, we aimed to dose of IVIG (Cao et al, 2020; Gharebaghi et al, 2020; Lanza et al, characterize how IVIG reduced inflammation in these individuals. 2020; Reynaga et al, 2020; Xie et al, 2020). Nevertheless, how IVIG Thus, we performed an analysis of 41 soluble biomarkers in plasma can help in the recovery of COVID-19 is not well understood. Here, by Luminex and ELISA (Table S1). Whereas most of these biomarkers we have shown that IVIG may reduce the levels of several soluble were not consistently altered during treatment (Figs S1 and S2), we factors involved in immune cell recruitment and activation. In have observed a descendant trend (P = 0.062, Wilcoxon matched- addition, IVIG could reduce the levels of C5a in plasma, a powerful pairs signed rank test) in the levels of some soluble proteins that inflammatory mediator, generated by C5 cleavage during com- might support the clinical benefit observed in IVIG-treated patients plement activation, which plays a major role recruiting and ac- (Figs 1 and 2). Unfortunately, the low number of IVIG-treated pa- tivating immune cells. tients did not allow us to find statistically significant differences. Figure 1. Th1, Th12, and Th17 biomarkers and sCD163 in plasma samples from IVIG-treated COVID-19+ individuals. (A, B, C, D) Levels of (A) TNF, macrophage inflammatory protein-1α, IL-6, and IL-8; (B) IL-17A and GM-CSF (C) sCD163 and (D) IL-5 and IL-10, were determined in plasma samples from high-dose IVIG-treated COVID-19+ individuals (n = 5) (IVIG group) by Luminex or ELISA at baseline (day 0) and days 3, 7, and 14 since treatment initiation. Publicly available data showing the levels of these molecules in plasma of IVIG-untreated COVID- 19 patients (control group) (n = 32) at enrollment (day 0) and days +3 and +7 were used for comparative purposes (https://www.olink.com/mgh-covid-study). Mean and standard error of the mean are shown. Data of IVIG group were analyzed using the Wilcoxon matched-pairs signed rank test. Data in the control group were analyzed using Friedman and Dunn’s multiple comparisons test. NPX, normalized protein expression. Dotted lines indicate the limit of detection. Anti-inflammatory effect of IVIG in COVID-19 patients Rodrı́guez de la Concepción et al. https://doi.org/10.26508/lsa.202001009 vol 4 | no 9 | e202001009 2 of 7 lung of pathogenic tissue-resident memory-like Th17 cells (Zhao et al, 2021). Interestingly, the levels of IL-17 and GM-CSF tended to decrease in IVIG-treated COVID-19 patients until day 7 since IVIG treatment initiation (Fig 1B). Although, IL-17A was hardly detected in the control group and no data of GM-CSF were available, the decreasing trend observed in IVIG-treated COVID-19 patients suggests that IVIG might modulate also the Th17 response (Maddur et al, 2011). However, no effect of IVIG treatment was observed on the levels of IFN-γ–related biomarkers (Fig S2). Despite IFN-γ was hardly detected in IVIG-treated patients, a reduction in the levels of CXCL- 10 was found in both IVIG-treated and control groups. In contrast, the levels of CXCL-9 did not change in any group (Fig S2). Soluble CD163 is considered a marker of monocyte/macrophage activation, and high levels of soluble CD163 have been linked to several pathologic conditions, such as infectious disease or au- toimmunity (Ingels et al, 2013). Accordingly, sCD163 is increased in plasma of SARS-CoV-2–infected individuals and has been associ- ated with the progression to severe COVID-19 (Gómez-Rial et al, 2020; Zingaropoli et al, 2021). Although IVIG-treated COVID-19 pa- tients showed an improvement on their clinical status that was associated with a decreasing trend in several biomarkers related to innate cells activation and recruitment, the levels of sCD163 showed a rapid increasing tendency (as early as 3 d after the first ad- ministration of IVIG) (Fig 1C). However, this increase was only ob- served at day 7 but not at day 3 in IVIG-untreated patients (Fig 1C). An increase in the plasmatic levels of sCD163 was also observed in a subgroup of patients affected by Kawasaki disease that positively responded to IVIG treatment (Azuma et al, 2020). Therefore, the Figure 2. Effect of IVIG treatment in the plasmatic levels of IL-7, hepatocyte levels of sCD163 observed in IVIG-treated COVID-19 patients might growth factor, fatty acid–binding protein 2, and C5a in COVID-19 patients. indicate an IVIG-dependent modulation of macrophage/monocyte (A, B, C) Levels of (A) IL-7, hepatocyte growth factor; (B) C5a complement factor; function and should not be considered as a biomarker of disease and (C) fatty acid–binding protein 2, and LPS-binding protein were determined in plasma samples from high-dose IVIG-treated COVID-19+ individuals (n = 5) (IVIG severity. group) by Luminex or ELISA at baseline (day 0) and days 3, 7 and 14 after In addition to proinflammatory cytokines and chemokines, the treatment initiation. Publicly available data showing the levels of these molecules Th2 cytokines IL-5 and IL-10 also showed a decreasing trend after in plasma of IVIG-untreated COVID-19 patients (control group) (n = 32) at the administration of high dose of IVIG (P = 0.062); however, the enrollment (day 0) and days +3 and +7 were used for comparative purposes (https://www.olink.com/mgh-covid-study). Mean and Standard Error of the Mean effect of IVIG treatment could not be confirmed because these are shown. Data of IVIG group were analyzed using the Wilcoxon matched-pairs cytokines were below the limit of detection in the untreated control signed rank test. Data in the control group were analyzed using Friedman and group (Fig 1D). Furthermore, the levels of IL-7 and hepatocyte Dunn’s multiple comparison test. NPX, normalized protein expression. Dotted growth factor (HGF), two key factors involved in lymphocyte gen- lines indicate the limit of detection. eration and expansion, and tissue remodeling and wound healing, respectively, also tended to decrease after IVIG treatment initiation Publicly available data describing the levels of these molecules (P = 0.062) (Fig 2A). These results contrasted with the dynamic of in plasma of COVID-19 patients were used for comparative pur- these proteins observed in untreated COVID-19 patients, who poses (https://www.olink.com/mgh-covid-study) (Filbin et al, 2021) showed an increase in the levels of those proteins over time. In- (Figs 1 and 2). Several proinflammatory factors, such as IL-6 (previ- terestingly, it has been described that HGF plays a major role in the ously reported [Reynaga et al, 2020]), IL-8, tumor necrosis factor expansion of myeloid-derived suppressor cells (Yen et al, 2013). (TNF), and macrophage inflammatory protein-1α (MIP-1α), showed a These cells show potent immunosuppressor properties, are ex- descendant trend in the IVIG-treated group (Fig 1A). However, the panded in severe COVID-19 patients, and their frequency correlate opposite behavior was observed for TNF and MIP-1α during the with the levels of HGF (Kvedaraite et al, 2021). Moreover, high levels natural course of the disease in COVID-19 patients (Fig 1A). The data of HGF have been associated with severity and mortality in SARS- also suggested that the IVIG might induce a faster decade in the CoV-2–infected individuals (Tamayo-Velasco et al., 2021). plasmatic levels of IL-6 and IL-8 (day 3 after treatment initiation) Complement activation has been also associated with tissue when compared with the reduction observed in the control group damage, hyperinflammation, and disease severity in COVID-19 in- (day 7) (Fig 1A). dividuals (Carvelli et al, 2020; Java et al, 2020; Shen et al, 2020). High In addition, high levels of IL-17A and GM-CSF have been de- levels of C5a have been associated with the severity of the disease scribed in both sera and bronchoalveolar fluid of severe COVID-19 in COVID-19 patients (Carvelli et al, 2020; Cugno et al, 2020), and patients, and their levels were associated with the expansion in the several therapies aimed to reduce complement activation are in Anti-inflammatory effect of IVIG in COVID-19 patients Rodrı́guez de la Concepción et al. https://doi.org/10.26508/lsa.202001009 vol 4 | no 9 | e202001009 3 of 7 clinical trials (Polycarpou et al, 2020). Furthermore, other highly path- complex with CD14 that finally induces the endocytosis and signaling ogenic viruses, such as H5N1, H7N9, SARS-CoV, or MERS-CoV, might also through TLR4 (Tsukamoto et al, 2018), promoting a proinflammatory induce acute lung injury by an excessive and uncontrolled complement cascade. It has been reported that the spike glycoprotein of SARS-CoV-2 activation (Wang et al, 2015). Here, we have shown that the levels of C5a can bind to LPS and promote inflammation in vitro and in vivo (Petruk et showed a descendant trend in plasma after treatment initiation (P = al, 2020). Moreover, the exposure of pigs to both porcine respiratory 0.062) (Fig 2B), suggesting that IVIG may reduce the levels of C5a in coronavirus and LPS enhanced respiratory disease by increasing the COVID-19 subjects. Although we do not have access to data from un- proinflammatory response (Van Reeth et al, 2000). Therefore, it is treated COVID-19 patients to confirm this descendant trend, it has been plausible to think that the LPS pathway might be involved in the de- reported that the levels of C5a remains stable at least during 10 d after velopment of the severe COVID-19 (Kruglikov & Scherer, 2021) and that disease initiation (Carvelli et al, 2020). In fact, among the different im- IVIG treatment might modulate this pathway. Nevertheless, no major munomodulatory mechanisms linked to high-dose IVIG treatment, the effect on soluble CD14 was observed (Fig S1), which remained at normal binding and blocking of C5a and C3a have been well documented (Basta levels in IVIG-treated individuals. et al, 2003). Therefore, the trend observed in the levels of C5a in the IVIG- The present work shows two main limitations: the low number of treated group is, at some extend, expected, and might be considered as a individuals analyzed and the lack of specific controls. To overcome the positive control of the immunomodulatory effect of the IVIG in COVID-19 later limitation, we have used publicly available data obtained from patients. Interestingly, we did not observe a reduction in the levels of COVID-19 patients. We have selected individuals that showed a similar sC5b-9 (Fig S1), suggesting that the formation of the membrane attack disease profile than the IVIG group. Therefore, our results suggest that complex might not be affected. Similar results were observed when high-dose IVIG administration may be a promising treatment for, at least, a specific reagents for blocking C5a or its receptor (C5aR1) were used subset of COVID-19–affected individuals. However, because of the limi- (Carvelli et al, 2020). C5a is a potent proinflammatory and chemo- tations described before, the results presented here need to be confirmed attractant factor for neutrophils, monocytes, and macrophages (Guo & in future studies. A randomized clinical trial is currently ongoing to Ward, 2005). C5a increases the production of TNF, MIP-1α, and IL-6 by corroborate our preliminary finding (clinical trial.gov: NCT04432324). monocytes isolated from COVID-19 patients and stimulated with LPS or R848 (Carvelli et al, 2020). Interestingly, all these proteins tend to be reduced in IVIG-treated COVID-19 patients. Overall, our results suggest that IVIG could decrease the hyper- Materials and Methods inflammation stage observed in SARS-CoV-2–infected individuals by Samples reducing the levels of cytokines, chemokines and some complement factors (C5a) involved in immune activation and cell migration. In- Blood samples were collected in EDTA tubes from five COVID-19 patients terestingly, most of the biomarkers described in the present work successfully treated with a 5-d course of 400 mg/kg/d of IVIG (Flebo- had been reported to be elevated in severe COVID-19 individuals gamma 10% DIF; Instituto Grifols). Blood samples were obtained at four (Blanco-Melo et al, 2020; Huang et al, 2020). different time points: Day 0 (pretreatment initiation), 3, 7, and 14 after the However, IVIG are multifunctional and they might also act through first administration of IVIG. Blood samples were processed within 3 h of additional mechanisms that need further investigation. In this sense, we collection. To collect plasma, blood samples were centrifuged at 3,000g have also observed a progressive reduction in the levels of fatty for 10 min at room temperature. Plasma samples were stored at −80°C acid–binding protein 2 (FABP-2) and LPS-binding protein (LBP) after IVIG until use. Clinical and demographic features of patients have been treatment initiation (Fig 2C). Plasmatic FABP-2 has been considered as a previously reported (Reynaga et al, 2020). Briefly, patients showed a biomarker associated with gut damage (Pelsers et al, 2005), and a multilobar pneumonia and PaO2/FiO2 values ≤300 mmHg. All required plasmatic increase in the levels of endotoxin (1 → 3)-β-d-glucan and supplemental oxygen therapy and showed a medium risk of acute acute bacterial DNA (markers of gut leakage) have been also described in respiratory distress syndrome according to the Berlin score. SARS-CoV-2 severe COVID-19 patients (Sirivongrangson et al, 2020). About 12% of infection was confirmed by RT-PCR in oropharyngeal swab samples (four COVID-19 patients manifest gastrointestinal symptoms whereas SARS- out of five patients) or by serology (one out of five patients). Their ages CoV-2 shedding in feces is observed in 40% of infected individuals were in the range of 24–80 yr and two of them were female. The study (Parasa et al, 2020). Therefore, the decreasing trend observed in FABP-2 was revised and approved by the Pharmacy and Therapeutics Com- and LBP might indicate that high-dose IVIG might reduce gut damage in mittee of the “Hospital Germans Trias I Pujol” (code: PI-20-266) and COVID-19 patients. However, this finding needs further investigation to conformed to the principles set out in the WMA Declaration of Helsinki. A clarify the role of these biomarkers in the pathogenesis of COVID-19 and signed informed consent was obtained for all participants. how high-dose IVIG treatment might modulate their functions. Guedj K et al have recently reported that the levels of FABP2 are lower in COVID- 19 patients than in uninfected individuals, and are not associated with Control group prognosis (Guedj et al, 2021). Moreover, despite it had been shown that COVID-19 patients may have high levels of LBP in serum (Hoel et al, 2020), Publicly available longitudinal data from COVID-19 patients were the plasmatic levels of this molecule are reduced in both IVIG-treated used as controls (https://www.olink.com/mgh-covid-study) (Filbin and untreated COVID-19 patients over time (Fig 2C). In spite of that, IVIG- et al, 2021). The control group (n = 32) was selected according to the treated individuals might show a faster decreasing trend because it was following criteria: COVID status: positive; age cat: 1 (20–34 yr old), 2 observed as early as day 3 compared with day 7 in the control group (Fig (36–49), 4 (65–79), and 5 (>80); respiratory symptoms: positive (sore 2C). LBP plays a major role in LPS signaling. It binds to LPS and forms a throat, congestion, productive or dry cough, shortness of breath or Anti-inflammatory effect of IVIG in COVID-19 patients Rodrı́guez de la Concepción et al. https://doi.org/10.26508/lsa.202001009 vol 4 | no 9 | e202001009 4 of 7 hypoxia, or chest pain); World Health Organization score basal 0: 4 C Avila-Nieto: data curation, investigation, methodology, and wri- (hospitalized, supplementary O2 required); C-reactive protein at ting—review and editing. day 0: 3 (0–19.9), 4 (100–179), or 5 (>180). Only individuals with data at JR Santos: investigation and writing—review and editing. day 0, 3, and 7 were considered. S Roure: investigation and writing—review and editing. L Mateu: investigation and writing—review and editing. Determination of biomarkers in plasma sample R Paredes: investigation and writing—review and editing. J Puig: data curation, investigation, and writing—review and editing. The concentration of several soluble biomarkers was determined in JM Jimenez: data curation and writing—review and editing. plasma samples by Luminex or ELISA. A description of the molecules N Izquierdo-Useros: investigation and writing—review and editing. determined, suppliers and kits used for the determination are showed B Clotet: conceptualization, supervision, funding acquisition, in- in Table S1. All determinations were performed following the manu- vestigation, visualization, and writing—review and editing. facturers’ instructions, except for the incubation of samples in ELISA ML Pedro-Botet: conceptualization, data curation, investigation, assays, which was performed at 4°C, overnight. For ELISA development, and writing—review and editing. the supersensitive 3, 39, 5, 59-tetramethylbenzidine (TMB) Liquid Sub- J Carrillo: data curation, formal analysis, supervision, validation, strate (T4444; Sigma-Aldrich) was used. Reaction was stopped using 2 M investigation, methodology, and writing—original draft. of H2SO4 and signal was analyzed as 450/620 ratio. Conflict of Interest Statement Statistical analysis B Clotet is the founder of AlbaJuna Therapeutics SL. J Carrillo is the cofounder The levels of each biomarker at day 0, 3, 7, or 14 in the IVIG group were and Chief Scientific Officer of AlbaJuna Therapeutics SL. B Clotet, J Carrillo, analyzed using the Wilcoxon matched-pairs signed rank test. The control and N Izquierdo-Useros are part of the CBIG consortium that is founded by group was analyzed using Friedman and Dunn’s multiple comparison Grifols. The authors declare no other conflict of interests. tests. Statistical analyses were conducted using GraphPad Prism v 7.0. 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