Circulating calprotectin levels four months after severe and non-severe COVID-19.

BACKGROUND: Calprotectin is an inflammatory marker mainly released by activated neutrophils that is increased in acute severe COVID-19. After initial recovery, some patients have persistent respiratory impairment with reduced diffusion capacity of the lungs for carbon monoxide (DLCO) months after infection. Underlying causes of this persistent impairment are unclear. We aimed to investigate the correlation between circulating calprotectin, persistent lung functional impairment and intensive care unit (ICU) stay after COVID-19 in two university hospital centres in Switzerland. METHODS: Calprotectin levels were measured in serum from 124 patients (50% male) from the Bern cohort (post-ICU and non-ICU patients) and 68 (76% male) from the Lausanne cohort (only post-ICU patients) four months after COVID-19. Calprotectin was correlated with clinical parameters. Multivariate linear regression (MLR) was performed to evaluate the independent association of calprotectin in different models. RESULTS: Overall, we found that post-ICU patients, compared to non-ICU, were significantly older (age 59.4 ± 13.6 (Bern), 60.5 ± 12.0 (Lausanne) vs. 48.8 ± 13.4 years) and more obese (BMI 28.6 ± 4.5 and 29.1 ± 5.3 vs. 25.2 ± 6.0 kg/m2, respectively). 48% of patients from Lausanne and 44% of the post-ICU Bern cohort had arterial hypertension as a pre-existing comorbidity vs. only 10% in non-ICU patients. Four months after COVID-19 infection, DLCO was lower in post-ICU patients (75.96 ± 19.05% predicted Bern, 71.11 ± 18.50% Lausanne) compared to non-ICU (97.79 ± 21.70% predicted, p < 0.01). The post-ICU cohort in Lausanne had similar calprotectin levels when compared to the cohort in Bern (Bern 2.74 ± 1.15 µg/ml, Lausanne 2.49 ± 1.13 µg/ml vs. non-ICU 1.86 ± 1.02 µg/ml; p-value < 0.01). Calprotectin correlated negatively with DLCO (r= -0.290, p < 0.001) and the forced vital capacity (FVC) (r= -0.311, p < 0.001). CONCLUSIONS: Serum calprotectin is elevated in post-ICU patients in two independent cohorts and higher compared to non-ICU patients four months after COVID-19. In addition, there is a negative correlation between calprotectin levels and DLCO or FVC. The relationship between inflammation and lung functional impairment needs further investigations. TRIAL REGISTRATION: NCT04581135.

Monocytes enhance neutrophil-induced blister formation in an ex vivo model of bullous pemphigoid.

BACKGROUND: Lesions of bullous pemphigoid (BP), an autoimmune subepidermal blistering disease characterized by the presence of tissue-bound and circulating autoantibodies to hemidesmosomal antigens, harbor a mixed inflammatory cellular infiltrate. In various models, neutrophils, eosinophils, mast cells, monocytes as well as B and T cells have been shown to be involved in the pathogenesis of BP. However, their interactions with and effective role in blister formation remain uncertain. This study was aimed at investigating the effect of monocyte/neutrophil interaction on blister formation in an ex vivo BP model. METHODS: Skin cryosections were incubated with purified human neutrophils and monocytes, in the presence or absence of BP autoantibodies. Production of reactive oxygen species (ROS), degranulation, mediator release (neutrophil elastase [NE], myeloperoxidase [MPO], matrix metalloproteinase-9 [MMP-9]), binding of Fcγ receptor (CD16, CD32, CD64), and cell adhesion (CD18, ICAM-1) was investigated using appropriate inhibitors. Dermal-epidermal separation (DES) was assessed by light microscopy and quantified by Fiji software. RESULTS: Monocytes and neutrophils synergistically interact resulting in a significantly higher DES compared to either monocytes or neutrophils separately (P < .0001). Monocyte/neutrophil-induced DES was associated with increased ROS production and was dependent on adhesion and FcγRIII binding. Upon stimulation by the granule-poor fraction of monocyte supernatants, neutrophils increased their release of MMP-9, thereby also DES at the dermal-epidermal junction of skin cryosections. CONCLUSION: Our observations suggest that the interaction of cells, as shown here for monocytes and neutrophils, enhances mediator release resulting in an increased subepidermal blister formation. Thus, blocking intercellular cross talk promises a new therapeutic approach for blocking tissue damage in BP.

Increased level of antibodies cross-reacting with Ves v 5 and CRISP-2 in MAR-positive patients.

BACKGROUND: Anti-sperm antibodies (ASA) have been described to be involved in immunological infertility. A possible antigen for ASA is the human cysteine-rich secretory protein 2 (CRISP-2), a sperm surface protein important in sperm-oocyte interaction. Furthermore, anti-CRISP-2 antibodies were shown to decrease fertility rates in vitro. Recently, we have reported cross-reacting antibodies recognizing CRISP-2 and antigen 5 from yellow jacket venom (Ves v 5) in human serum. METHODS: Here, we investigated anti-Ves v 5 and CRISP-2 antibodies in sera from two groups of donors: MAR+ and MAR- patients. RESULTS: A higher incidence of allergy against hymenoptera venom was found in MAR+ patients. Interestingly, affinity-purified ASA from MAR+ patients' sera reacted against both Ves v 5 and CRISP-2, leading to sperm immobilization. Immunofluorescence analysis showed that ASA bound to the sperm surface, including the head part where CRISP-2 is localized. CONCLUSION: Taken together, these results showed a higher incidence of antibodies cross-reacting with Ves v 5 and CRISP-2 in MAR+ patients. This leads to the hypothesis that MAR+ patients may have a higher risk to develop wasp allergy.

Conditional autoimmunity mediated by human natural anti-Fc(epsilon)RIalpha autoantibodies?

Natural antibodies provide an early defense mechanism against pathogens, show a frequent self-reactivity, and are present throughout life. Two questions concern the physiological control of self-reactivity and the pathogenetic link to autoimmune disease. Here we propose a concept of conditional autoimmunity involving natural antibodies against the alpha chain of the high-affinity receptor for IgE (Fc(epsilon)RIalpha ). Like other natural antibodies, anti-Fc(epsilon)RIalpha antibodies are found in sera of healthy donors. We now report the first human recombinant anti-Fc(epsilon)RIalpha autoantibodies isolated by repertoire cloning from a human tonsillar IgM library. These high-affinity antibodies recognize Fc(epsilon)RIalpha on cells and trigger histamine release from freshly isolated blood basophils. However, the latter effect requires IgE removal from the Fc(epsilon)RI. The same conditional histamine release is seen when using sera from individual normal donors and affinity-purified anti-Fc(epsilon)RIalpha antibodies isolated from multidonor therapeutic IgG preparations. We propose that such anti-Fc(epsilon)RIalpha antibodies can become pathogenic and that this is dependent on the state of occupancy of the Fc(epsilon)RIalpha by its natural ligand IgE. We suggest that an imbalance between Fc(epsilon)RIalpha occupancy and natural anti-Fc(epsilon)RIalpha antibodies may be implicated in the pathogenesis of autoimmune urticaria.

Natural anti-FcepsilonRIalpha autoantibodies isolated from healthy donors and chronic idiopathic urticaria patients reveal a restricted repertoire and autoreactivity on human basophils.

The role of autoantibodies against the alpha-subunit of the human high-affinity IgE receptor (FcepsilonRIalpha) in the pathogenesis of chronic idiopathic urticaria (CIU) is controversial. We have shown that these antibodies are widespread, apparently non-pathogenic and belong to the natural antibody repertoire. To clarify this controversy, we constructed antibody libraries from both healthy donors and CIU patients with active disease. Here we describe the first three high affinity IgM anti-FcepsilonRIalphaautoantibodies isolated from normal and urticaria libraries. Sequence analysis revealed germline VH in both cases paired with a slightly mutated VL, thus supporting their classification as natural antibodies. Strikingly, one major IgM clone was present in both CIU patients and normal donors. The anti-FcepsilonRIalpha autoantibodies recognize FcepsilonRIalpha on cells, but are non-anaphylactogenic on blood basophils, except when IgE is removed from the receptor. Based on their functional activities we propose a concept of "conditional autoimmunity" where natural anti-FcepsilonRIalphaautoantibodies can become pathogenic dependent on the state of occupancy of the FcepsilonRIalpha by its natural ligand IgE.

Human anti-FcepsilonRIalpha autoantibodies isolated from healthy donors cross-react with tetanus toxoid.

Natural antibodies (Ab) reacting with self antigens have been shown to be present in all individuals. These autoantibodies (auto-Ab) can be either pathogenic or non-pathogenic. Auto-Ab reacting with the alpha-subunit of the high-affinity receptor for IgE (FcepsilonRIalpha) have been implicated in the pathogenesis of a subset of patients with chronic idiopathic urticaria (CIU). Intravenous immunoglobulin (IVIg) preparations have been used with variable clinical benefit in the treatment of these patients. Here we show that anti-FcepsilonRIalpha auto-Ab are present in a therapeutic IVIg preparation as well as in atopic and chronic urticaria patients and healthy individuals. We affinity-purified the anti-FcepsilonRIalpha Ab from an IVIg preparation using recombinant FcepsilonRIalpha. Interestingly, these anti-FcepsilonRIalpha auto-Ab showed no evidence of histamine release but strongly cross-reacted with an external antigen, tetanus toxoid (TTd) with a higher affinity for TTd than for the FcepsilonRIalpha. Since the cross-reacting Ab are non-anaphylactogenic, there is no evidence that TTd immunization may contribute to the pathogenesis of CIU. However, our results may indicate that the anti-FcepsilonRIalpha auto-Ab belong to the natural Ab and serve as the parental Ab for some anti-TTd Ab.